Deoxo-proline-containing tamandarin and didemnin analogs, dehydro-proline-containing tamandarin and didemnin analogs, and methods of making and using them

ABSTRACT

The present invention relates to tamandarin and didemnin analogs which have a deoxo-proline residue or a dehydro-proline residue in their structure. These analogs are useful as anti-cancer agents and for other purposes. Methods of making these analogs and methods of using them as inhibitors of protein synthesis, cell growth, and tumorigenesis and as enhancers of apoptosis are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 09/545,848, which was filed on Apr. 7, 2000.

STATEMENT REGARDING FEDERALLY SUPPORTED RESEARCH OR DEVELOPMENT

This research was supported in part by U.S. Government funds (NIH grantnumber CA40081), and the U.S. Government may therefore have certainrights in the invention.

BACKGROUND OF THE INVENTION

Didemnin B is a macrocyclic depsipeptide isolated from a species ofmarine tunicate. Didemnin B exhibits potent anti-viral,immunosuppressive, and anti-tumor activities in vitro and in vivo, andwas the first marine natural product to enter clinical testing againsthuman cancers (Li et al., 1992, Studies in Natural Products Chemistry,10:241-302; Sakai et al., 1996, J. Med. Chem. 39:2819-2834; Wipf, 1995,Chem. Rev. 95:2115-2134). Didemnin B is a didemnin, a family ofcompounds which potently inhibit protein synthesis and cell cycleprogression, and induce more rapid apoptosis than any other naturalproducts that has been isolated to date (Grubb et al., 1995, Biochem.Biophys, Res. Commun. 215:1130-1136; Johnson et al., 1996, FEBS Lett.383:1-5; Johnson et al., 1999, Immunol. Cell Biol. 77:242-248; Johnsonet al., 1999, J. Cell. Biochem. 72:269-278). Other members of thisfamily of compounds, including didemnin M and dehydrodidemnin B, exhibitcytotoxic and cytostatic effects as well.

Tamandarin A (also designated {(2S)Hiv²}didemnin B) is a naturallyoccurring didemnin congener which has recently been isolated from amarine tunicate. Tamandarin A exhibits biological activity which isanalogous to the activities exhibited didemnin B. For example,tamandarin A is a potent inhibitor of protein synthesis, cell growth,and tumorigenesis. Tamandarin A exhibits greater in vitro activityagainst pancreatic carcinoma than does didemnin B (Liang et al., 1999,Org. Lett. 1: 1319-1322). A significant limitation on use of tamandarinA, either for research or for practical applications, is the limitedsupply of tamandarin A that is available from natural sources and thedifficulty and expense of isolating this product. A need exists for amethod of synthesizing tamandarin A and other didemnin analogs(including dehydrodidemnin analogs).

Despite the potency of didemnin B in isolated studies, its clinicaleffectiveness is hampered by side effects associated with therapeuticdoses of the compound. As with many anti-proliferative agents, didemninB exhibits a relatively narrow therapeutic window. Although didemnin Mand dehydrodidemnin B exhibit improved therapeutic potential, relativeto didemnin B, a need still exists for anti-proliferative agents whichexhibit less toxicity at a therapeutic dose (i.e. didemnin analogshaving a greater therapeutic index).

The present invention satisfies the needs set forth above.

BRIEF SUMMARY OF THE INVENTION

The invention relates to tamandarin and didemnin analogs that have adeoxo-proline residue or a dehydro-proline residue in their structure.In one embodiment, the invention relates to a composition comprising atamandarin analog having the structure of formula I

In formula I, R is selected from the group consisting of

-   —(N-methyl)leucine-deoxo-proline,-   —(N-methyl)leucine-deoxo-proline-lactate,-   —(N-methyl)leucine-deoxo-proline-pyruvate,-   —(N-methyl)leucine-deoxo-proline-lactate-(a first fluorophore),-   —(N-methyl)leucine-deoxo-proline-lactate-glutamine-pyroglutamate,-   —(N-methyl)leucine-deoxo-proline-lactate-glutamine-cyclopentanoate,-   —(N-methyl)leucine-deoxo-proline-alanine-leucine-pyroglutamate, and-   —(N-methyl)leucine-deoxo-proline-(N-methyl-alanine)-leucine-pyroglutamate,-   —(N-methyl)leucine-dehydro-proline,-   —(N-methyl)leucine-dehydro-proline-lactate,-   —(N-methyl)leucine-dehydro-proline-pyruvate,-   —(N-methyl)leucine-dehydro-proline-lactate-(a first fluorophore),-   —(N-methyl)leucine-dehydro-proline-lactate-glutamine-pyroglutamate,-   —(N-methyl)leucine-dehydro-proline-lactate-glutamine-cyclopentanoate,-   —(N-methyl)leucine-dehydro-proline-alanine-leucine-pyroglutamate,    and-   —(N-methyl)leucine-dehydro-proline-(N-methyl-alanine)-leucine-pyroglutamate.

R² and R³ in formula I, can be separate moieties or they can, together,be a single moiety. When R² and R³ are separate moieties, R³ is either amethyl group or a hydride radical and R² is selected from the groupconsisting of an isoleucine side chain, a valine side chain, an alanineside chain, a norleucine side chain, a norvaline side chain, leucineside chain, a histidine side chain, a tryptophan side chain, an arginineside chain, a lysine side chain, a second fluorophore, and a substituenthaving the structure of formula III

When R² and R³ are, together, a single substituent, this substituent hasthe structure of formula IV

In formulas III and IV, each of R⁵, R⁶, R⁷, R⁸, and R⁹ is independentlyselected from the group consisting of —H, —OH, —OCH₃, —CO(C₆H₅), —Br,—I, —F, —Cl, —CH₃, and —C₂H₅.

R⁴ in formula I is either an isoleucine side chain or a valine sidechain. Also, in formula I, X is either —O— or —(NH)—, Y is either ahydride radical or a hydroxyl protecting group, and R¹⁰ is either aleucine side chain or a lysine side chain. The didemnin analog is ananalog other than tamandarin A (i.e. {(2S)Hiv² }didemnin B). In oneembodiment, every proline or lactate moiety that is present in R¹exhibits (S) stereochemistry. In another, every moiety capable ofexhibiting stereochemistry in R¹ is present in its naturally occurringform (i.e. the (S) form for amino acid residues and lactate. It isbelieved that cyclopentanoate occurs naturally in an (S)stereochemistry.

In another embodiment, the invention relates to a composition comprisinga didemnin analog having the structure of formula XXI

In formula XXI, each of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ hasthe same meaning as in formula I.

In preferred classes of deoxo-proline tamandarin and didemnin analogshaving the formula of I and XXI, respectively, R² has the structure offormula III, R³ is methyl, R⁴ is an isoleucine side chain, each of R⁵,R⁶, R⁸, and R⁹ is a hydride radical, R⁷ is methoxy, R¹⁰ is a leucineside chain, X is —O—, and Y is a hydride radical. Examples of tamandarinand didemnin analogs that are included in the invention are compounds201, 202, 203, and 204, which are shown in FIGS. 1, 2, 7, and 8respectively.

In one embodiment, the tamandarin or didemnin analog has a photoreactivesubstituent, such as an R² moiety having the structure

In another embodiment, the tamandarin or didemnin analog has afluorophore attached, such as an analog in which a fluorophore isattached at the omega amino moiety of a lysine side chain at R² or atR¹⁰. Alternatively, the didemnin analog can be attached (e.g.covalently) with a support. In most embodiments, Y in formulas I and XXIis preferably a hydride radical.

The invention includes an embodiment of a tamandarin or didemnin analogwhich can be activated (or the activity of which can be enhanced) byenzymatic cleavage of a moiety bound with the analog. For example, theinvention includes compositions which comprise a tamandarin analoghaving a structure selected from the group consisting of formulas(a)-(d), as follows.

In formulas (a)-(d), R², R³, R⁴, R¹⁰, X, and Y have the same identitiesdescribed above for formula I. R¹³ is an enzyme-cleavable moiety that iscleavable by an enzyme, such as one selected from the group consistingof a carboxypeptidase, a beta-lactamase, a beta-galactosidase, apenicillin V-amidase, a cytosine deaminase, a nitroreductase, a alkalinephosphatase, a beta-glucuronidase, and a catalytic antibody. By way ofexample, R¹³ can have the structure of either of formulas V and VI

The tamandarin and didemnin analogs described herein can be formulated,together with one or more pharmaceutically acceptable carriers, to makepharmaceutical preparations. These preparations can be administered to amammalian (e.g. human) cell (i.e. either in vitro or in vivo) in orderto inhibit protein synthesis, inhibit growth, inhibit proliferation,inhibit tumorigenesis, or enhance apoptosis in the cell or in one ormore tissues of the mammal.

The invention further relates to a method of makingdeoxo-proline-containing tamandarin and didemnin analogs. These methodsemploy known methods for making tamandarin and didemnin analogs, and aremodified to incorporate a deoxo-proline residue in place of a prolineresidue of the analog.

The invention still further relates to a method of makingdehydro-proline-containing tamandarin and didemnin analogs. Thesemethods employ known methods for making tamandarin and didemnin analogs,and are modified to incorporate a dehydro-proline residue in place of aproline residue of the analog.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the structure of a preferred deoxo-proline tamandarin analogdesignated compound 201.

FIG. 2 is the structure of a preferred deoxo-proline didemnin analogdesignated compound 202.

Each of FIG. 3, FIG. 4, and FIG. 5 depicts a method of makingdeoxo-proline-containing side chain moieties for tamandarin or didemninanalogs.

FIG. 6 depicts a method of making dehydro-proline-containing side chainmoieties for tamandarin or didemnin analogs.

FIG. 7 is the structure of a preferred dehydro-proline tamandarin analogdesignated compound 203.

FIG. 8 is the structure of a preferred dehydro-proline didemnin analogdesignated compound 204.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to tamandarin and didemnin analogs which have adeoxo-proline residue or a dehydro-proline residue in their structure.The invention includes compositions comprising these deoxo-proline ordehydro-proline tamandarin and didemnin analogs, and methods for makingand using these analogs. These analogs are useful for, among otherthings, inhibiting protein synthesis, cell growth, cell proliferation,and tumorigenesis. The analogs of the invention can also exhibitanti-viral, anti-tumor, apoptosis-inducing, and immunosuppressiveactivities in animals, including in humans.

The invention includes compositions comprising a tamandarin analoghaving the structure

wherein R¹, R², R³, R⁴, R¹⁰, X, and Y have the identities describedherein.

The invention also includes compositions comprising a didemnin analoghaving the structure

wherein R¹, R², R³, R⁴, R¹⁰, X and Y have the identities describedherein.

DEFINITIONS

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

As used herein, amino acid residues are represented by the full namethereof, by the three letter code corresponding thereto, or by theone-letter code corresponding thereto, as indicated by the following:Full Name Three-Letter Code One-Letter Code Aspartic Acid Asp D GlutamicAcid Glu E Lysine Lys K Arginine Arg R Histidine His H Tyrosine Tyr YCysteine Cys C Asparagine Asn N Glutamine Gln Q Serine Ser S ThreonineThr T Glycine Gly G Alanine Ala A Valine Val V Leucine Leu L IsoleucineIle I Methionine Met M Proline Pro P Phenylalanine Phe F Tryptophan TrpW

As used herein, the term “amino acid side chain” refers to a moietycomprising all of the atoms of an amino acid excluding the alpha-carbonatom, a hydrogen atom bound with the alpha-carbon, the atoms of thealpha-carboxyl moiety and the alpha-amine moiety. By way of example, an“alanine side chain” refers to a methyl group, and a “valine side chain”refers to a 2-propyl group.

“Inhibition” of a process in a cell (e.g. inhibition of proteinsynthesis, inhibition of cell growth, inhibition of cell cycleprogression, inhibition of cell proliferation, or inhibition oftumorigenesis) means reduction (e.g. by at least 10%, 25%, 50%, 75%,90%, 95%, or even 100%) of the rate at which the process proceeds,reduction (e.g. by at least 10%, 25%, 50%, 75%, 90%, 95%, or even 100%)of the rate at which the process is initiated, or both.

“Enhancement” of a process in a cell (e.g. enhancement of apoptosis)means increasing (e.g. by at least 10%, 25%, 50%, 75%, 90%, 95%, or even100%) the rate at which the process proceeds, increasing (e.g. by atleast 10%, 25%, 50%, 75%, 90%, 95%, or even 100%) the rate at which theprocess is initiated, or both.

As used herein, the term “pharmaceutically acceptable carrier” means achemical composition with which a didemnin analog or fragment, asdescribed herein, can be combined and which, following the combination,can be administered to a subject (e.g. a human or other animal).

As used herein, the term “physiologically acceptable” ester or saltmeans an ester or salt form of a didemnin analog or fragment, asdescribed herein, which is compatible with other ingredients of apharmaceutical composition and which is not deleterious to a subject towhich the composition is to be administered.

As used herein, “parenteral administration” of a pharmaceuticalcomposition includes any route of administration characterized byphysical breaching of a tissue of a subject and administration of thepharmaceutical composition through the breach in the tissue. Parenteraladministration thus includes, but is not limited to, administration of apharmaceutical composition by injection of the composition, byapplication of the composition through a surgical incision, byapplication of the composition through a tissue-penetrating non-surgicalwound, and the like. In particular, parenteral administration caninclude, but is not limited to, subcutaneous, intraperitoneal,intramuscular, intrasternal injection, and kidney dialytic infusiontechniques.

As used herein, the term “anti-viral activity” means preventingreplication of a virus in the cell, preventing infection of the cell bya virus, or reversing a physiological effect of infection of the cell bya virus. An anti-viral agent a composition of matter which, whendelivered to a cell, exhibits anti-viral activities. Anti-viral agentsare well known and described in the literature. By way of example, AZT(zidovudine, Retrovir® Glaxo Wellcome Inc., Research Triangle Park,N.C.) is an anti-viral agent which is thought to prevent replication ofHIV in human cells.

As used herein, a “deoxo-proline” moiety or residue is a chemical moietywhich has the following structure.

As used herein, a “dehydro-proline” moiety or residue is a chemicalmoiety which has the following structure.

DESCRIPTION

The present invention relates to tamandarin and didemnin analogs havinga deoxo-proline moiety or a dehydro-proline in their structure. Theseanalogs exhibit potent pharmacological properties when administered tohumans and other mammals. By way of example, these compounds can inhibitprotein synthesis and cell growth and proliferation. These compounds canalso enhance apoptosis in cells. These properties render the compoundsuseful for treating a variety of disorders which are characterized byone or more of aberrant protein synthesis, aberrant cell growth,aberrant proliferation of cells, and aberrant apoptosis. Examples ofsuch disorders include tumorigenesis, tumor growth, tumor metastasis,infection of a cell by a virus, replication of a virus within a cell.

Among the compositions of the inventions are those which comprise atamandarin analog having the structure of formula I or a didemnin analoghaving the structure of formula XXI.

The R substituent of formulas I and XXI has a deoxo-proline moiety inits structure, and can, for example, be a polypeptide comprising one ormore amino acid residues in addition to the deoxo-proline residue.Examples of such polypeptides include

-   —(N-methyl)leucine-deoxo-proline,-   —(N-methyl)leucine-deoxo-proline-lactate,-   —(N-methyl)leucine-deoxo-proline-pyruvate,-   —(N-methyl)leucine-deoxo-proline-lactate-glutamine-pyroglutamate,-   —(N-methyl)leucine-deoxo-proline-lactate-glutamine-cyclopentanoate,-   —(N-methyl)leucine-deoxo-proline-lactate-leucine-pyroglutamate,-   —(N-methyl)leucine-deoxo-proline-alanine-leucine-pyroglutamate, and-   —(N-methyl)leucine-deoxo-proline-(N-methyl)alanine-leucine-pyroglutamate,-   —(N-methyl)leucine-dehydro-proline,-   —(N-methyl)leucine-dehydro-proline-lactate,-   —(N-methyl)leucine-dehydro-proline-pyruvate,-   —(N-methyl)leucine-dehydro-proline-lactate-(a first fluorophore),-   —(N-methyl)leucine-dehydro-proline-lactate-glutamine-pyroglutamate,-   —(N-methyl)leucine-dehydro-proline-lactate-glutamine-cyclopentanoate,-   —(N-methyl)leucine-dehydro-proline-alanine-leucine-pyroglutamate,    and-   —(N-methyl)leucine-dehydro-proline-(N-methyl-alanine)-leucine-pyroglutamate.

Additional examples of alternative R¹ substituents includedeoxo-proline-containing peptides which comprise a fluorophore (e.g.,rhodamine or coumarin), an enzymatically-cleavable group, or anotherchemical moiety bound (e.g. covalently attached) with a support (e.g. aglass or silica plate, an agarose or other polymeric bead, etc.). WhenR¹ comprises an N-methyl-leucine residue, the alpha-carbon atom of thatresidue can have either (R) or (S) stereochemistry. Other amino acidresidues within R¹ can have either (R) or (S) stereochemistry, but theypreferably have (S) stereochemistry at their alpha-carbon atom. When R¹comprises a lactate residue, the lactate residue is preferably an(S)lactate residue. In a preferable embodiment, every amino acid residuewithin R¹ other than the leucine (or N-methyl-leucine) residue (ifpresent) attached directly to the nitrogen atom of the ring of formula Ior XXI has (S) stereochemistry.

R³ can be either of —CH₃ and —H. Alternatively, R³ can, together withR², be a single substituent.

The R² substituent can be an amino acid side chain such as an isoleucineside chain (i.e. a 2-butyl moiety, preferably having (R)stereochemistry), a valine side chain (i.e. a 2-propyl moiety), analanine side chain (i.e. a methyl moiety), a norleucine side chain (i.e.a 1-butyl moiety), a norvaline side chain (i.e. a 1-propyl moiety), aleucine side chain (i.e. an isobutyl moiety, preferably having (S)stereochemistry), a phenylalanine side chain (i.e. a phenylmethylmoiety), a histidine side chain (i.e. a 4-methyl-imidazole moiety), atryptophan side chain (i.e. a 3-methyl-indole moiety), a tyrosine sidechain (i.e. a 4-hydroxy-phenylmethyl moiety), an arginine side chain(i.e. a 4-guanidinyl-butyl moiety), and a lysine side chain (i.e. a4-aminobutyl moiety).

An R² substituent can comprise a fluorophore (e.g. a fluorophore linkedwith one of the amino acid side chains described above). In addition, R²substituent can have the structure of formula III

In an alternative embodiment, R² and R³ together are a substituenthaving the structure of formula IV

In formulas III and IV, each of R⁵, R⁶, R⁷, R⁸, and R⁹, independently,can be a substituent selected from the group consisting of —H, —OH,—OCH₃, —CO(C₆H₅), —Br, —I, —F, —Cl, —CH₃, and —CH₂CH₃.

R⁴ can be an isoleucine side chain or a valine side chain.

X can be —H or —(NH)—.

Y can be —H or a hydroxyl protecting group. Examples of hydroxylprotecting groups which can be present at Y include an alkyl-substitutedsilyl moiety, an aryl-substituted silyl moiety, or a silane substitutedwith both alkyl- and aryl-moieties. An example of a useful hydroxylprotecting group is a triisopropylsilyl moiety. Other hydroxylprotecting groups which can be used at Y in formula I are described inreferences such as Green and Wutz (1999, Protecting Groups in OrganicSynthesis, Wiley, New York).

R¹⁰ can be an amino acid side chain such as a leucine side chain or alysine side chain. Alternatively, R¹⁰ can be an amino acid or otherchemical moiety which is bound with (e.g. covalently attached to) asupport (e.g. a solid support).

Another group of compositions included within the invention are thosewhich comprise a deoxo-proline tamandarin analog having a structureselected from the group consisting of formulas (a)-(d), set forth above.

Each of R², R³, R⁴, R¹⁰, X, and Y has the same meaning in formulas(a)-(d) that it has in formulas I and XXI.

In formulas (a)-(d), R¹³ can be hydrogen or a chemical moiety which canbe enzymatically cleavable (i.e. an enzyme-cleavable moiety). As usedherein, an enzyme-cleavable moiety can include any chemical moiety whichcan be cleaved (i.e. chemically detached from) in the presence of aspecific enzyme. Examples of enzymes capable of chemically detaching anenzyme-cleavable moiety include carboxypeptidases, beta-lactamase,beta-galactosidase, penicillin V-amidase, cytosine deaminase,nitroreductase, alkaline phosphatase, beta-glucuronidase, and catalyticantibodies. Examples of enzyme-cleavable moieties which can beincorporated in a compound described herein include cephalosporins,beta-glucosides, phosphate, pyrophosphate, beta-D-galactosides,nitrobenzamidine, cytosine, carbamates, peptides, and amino acids.Alternatively, R¹³ can be an enzyme-cleavable moiety such as adi-peptide linked with glutamine-pyroglutamate, or a moiety having thestructure of formula V or formula VI

After cleavage of an enzyme-cleavable moiety by an enzyme, the resultingdidemnin analog can exhibit one or more of the physiological activitiesdescribed herein. A tamandarin or didemnin analog having the structureof one of formulas (a)-(d), wherein R¹³ is an enzyme-cleavable moiety,can, optionally, exhibit these activities before the cleavage of theenzyme-cleavable moiety. However, in a preferred embodiment, the analogexhibits therapeutic activity only following cleavage of theenzyme-cleavable moiety therefrom.

As described above, a tamandarin or didemnin analog having the structureof one of formulas I, XXI, and (a)-(d) can be bound with a support. Theidentity of the support is not critical. The support can besubstantially any material with which such an analog can be bound (e.g.by covalent attachment through one of the R¹⁰ or R¹ moieties). Examplesof support materials include bonded silicates, cross-linked agarose,polyacrylamide, dextran, and allyl dextran. Such support materials canbe chemically modified using reactive chemical moieties in order tofacilitate covalent attachment of the analog with the support. Chemicalmodifications of this type are known in the art, and can, for example,include modification of a support with cyanogen bromide groups, epoxidegroups, mesyl groups, and carboxyhexyl groups. Protocols for preparationof a support and subsequent attachment of a compound to the support areavailable in the art, and can be modified by one skilled in the art foruse with a didemnin analog described herein.

Preferred tamandarin analogs are based on the structure of tamandarin A.In these analogs, R² is an O-methyl-tyrosine side chain (i.e. R⁵, R⁶,R⁸, and R⁹ are each —H, and R⁷ is —OCH₃), R³ is —CH₃, R⁴ is anisoleucine side chain, R¹⁰ is a leucine side chain, X is —O—, and Y is—H.

Preferred didemnin analogs are based on the structure of didemnin B. Inthese analogs, R² is an O-methyl-tyrosine side chain (i.e. R⁵, R⁶, R⁸,and R⁹ are each —H, and R⁷ is —OCH₃), R³ is —CH₃, R⁴ is an isoleucineside chain, R¹⁰ is a leucine side chain, X is —O—, and Y is —H.

Methods of Using Compounds Described Herein

Deoxo-proline-containing tamandarin and didemnin analogs anddehydro-proline-containing tamandarin and didemnin analogs, as each aredisclosed herein, can be used to affect a variety of physiologicalprocesses. Each of these types of compounds can be used to inhibitprotein synthesis. Furthermore, the compounds can be used to inhibitprogression of a cell through the cell cycle. While not being bound byany particular theory of operation, it is believed that the cellcycle-inhibiting activity of the compounds can be attributed toinhibition of protein synthesis and possibly also to inhibition of othercellular activities associated with DNA replication or cell division.These tamandarin and didemnin analogs also induce apoptosis in cells.The physiological activities attributable to these tamandarin anddidemnin analogs make these compounds useful for alleviating a varietyof disorders in which one or more of cell growth, proliferation, andsurvival are aberrant. Examples of such disorders include cancers atvarious stages (e.g. tumorigenesis, tumor growth, and metastasis) andviral infections at various stages (e.g. infection of cells with virusparticles, production of virus particles within a cell, and survival ofvirus-infected cells).

While still not being bound by any particular theory of operation, it isbelieved that the physiological activities attributable to thetamandarin and didemnin analogs described herein result from one or moreinteractions between such analogs and at least one cellular component.This interaction(s) leads, directly or indirectly, to the observedcellular response. Accordingly, the invention encompasses use of thesecompounds to identify one or more cellular components which contributesto a disorder phenotype in an individual. Identification of such acellular component can indicate an effective course of treatment foralleviating the disorder. Examples of compounds useful for this purposeinclude analogs which comprise a fluorescent substituent (e.g., at R¹ orR²), a photoreactive chemical moiety, such as a moiety having thestructure

or a moiety bound with a support.

Fluorescent and other detectably labeled deoxo-proline tamandarin anddidemnin analogs described herein (as well as their physiologicallyactive fragments) can be used to identify cells in which those analogsand fragments can exert their physiological effects. For example, cellswhich absorb or bind with a fluorescent analog can be identified orisolated. Identification or isolation of such cells can be used todiagnose a disorder associated with the presence of such cells.Identification or isolation of these cells can also indicate which ofthe tamandarin or didemnin analogs are efficacious for treating adisorder involving the cells.

The tamandarin and didemnin analogs described herein can be used foranti-proliferative, anti-tumor, anti-viral, and immunosuppressivepurposes. For example, these compounds can be used in a pharmaceuticalpreparation or medicament to be administered to a patient afflicted witha disorder in which one or more of protein synthesis, cell growth,proliferation, and survival are aberrant. Such medicaments can be usedto treat disorders such as cancers (e.g. breast cancer), viral, fungal,parasitic, and bacterial infections, auto-immune disorders, allergies,other hyper-immune disorders, and atherosclerosis.

Examples of anti-tumor activities that can be exhibited by the compoundsdescribed herein include inhibition of tumorigenesis, inhibition ofmetastasis, inhibition of tumor cell growth, inhibition of tumor cellproliferation, and enhancement of tumor cell apoptosis. Dehydrodidemninexhibits activity against cell lines derived from several human solidtumor types, including non-small cell lung cancer and colon tumor celllines, and exhibits selective anti-tumor activity against non-small celllung cancer, melanomas, ovarian cancer, and colorectal cancer(Depenbrock et al., 1998, Brit. J. of Cancer 78(6): 739-744). Thetamandarin and didemnin analogs described herein exhibit anti-tumoractivities in cells of one or more of these lines, as well as in cellsof the corresponding tumor type in vivo. Determination of theeffectiveness of any particular analog against any particular tumor typecan be made using standard methods involving, for example, one or moreof the 60 standard tumor cell lines maintained in the U.S. NationalCancer Institute drug screening program.

Examples of anti-viral activities that can be exhibited by thetamandarin and didemnin analogs described herein include inhibition ofbinding of a virus with a cellular target, inhibition of infection of acell by a virus, inhibition of cellular synthesis of virus components,inhibition of intracellular assembly of virus particles, inhibition ofrelease of virus particles from an infected cell, inhibition of growthof a cell infected by a virus, inhibition of proliferation of a cellinfected by a virus, and induction of death (i.e. apoptosis) of a cellinfected by a virus. The anti-viral activity of the compounds describedherein can, for example, be used to treat or prevent viral infections ofmammals and associated symptoms. By way of illustration, adeoxo-proline-containing tamandarin or didemnin analog, or adehydro-proline-containing tamandarin or didemnin analog, can be used totreat or prevent infections by viruses such as Rift Valley Fever virus,Dengue virus, or any of the equine encephalitis viruses.

Examples of immunosuppressive activities that can be exhibited by thetamandarin and didemnin analogs described herein include inhibition of acellular immune response to an immunogen (e.g. an infectious agent, or atransplanted cell or tissue) and inhibition of a humoral immune responseto an immunogen. Examples of disorders in which immunosupression can bedesirable include autoimmune disorders, transplant rejection disorders(e.g. rejection of a solid tissue or bone marrow transplant),development of an immune response to an implanted device (e.g. a stentor a heart valve), immune hypersensitivity, and anaphylaxis.

The tamandarin and didemnin analogs described herein can be administeredin vitro to a cell or tissue (e.g. a cultured cell or tissue, or a cellor tissue harvested from one animal prior to introduction into the sameor a different animal). Alternatively, the analogs can be administeredto the cell or tissue in vivo by administering the analog or apharmaceutical composition comprising the analog to an animal (e.g. amammal such as a human) that comprises the cell or tissue.

In one embodiment of the treatment methods described herein, atamandarin or didemnin analog described herein and having anenzyme-cleavable group attached thereto (e.g. a compound having thestructure of formula II) is administered to an animal. Upon cleavage ofthe enzyme-cleavable group, the compound is transformed from an inactive(or less active) form to an active (or more active) form. Thus, thedeoxo-proline tamandarin or didemnin analog can be selectively activatedat a body location at which the enzyme activity occurs.

The enzyme which is used to cleave a tamandarin or didemnin analoghaving an enzyme-cleavable moiety attached can be an enzyme whichnaturally occurs at a body location in an animal. Alternatively, theenzyme can be provided to the animal, for example as a compositioncomprising the enzyme or a nucleic acid which encodes the enzyme. Asanother example, the enzyme can be coupled (e.g. covalently, using across-linking agent or by expression as an enzyme-antibody fusionprotein) with an antibody that specifically binds with a tissue (e.g.cancerous cells such as leukemic cells or cells of a solid tumor) at abody location in the animal, and the antibody-enzyme complex can beadministered to an animal. Administration of a deoxo-proline tamandarinor didemnin analog having an attached enzyme-cleavable group to the sameanimal results in preferential activation of the compound at the tissueor body location. The physiological effect of the compound can therebybe localized at the tissue or body location, and any side effectattributable to the activated compound can thereby be reduced orminimized.

A support-bound tamandarin or didemnin analog can be used to identifycells which comprise, on their surfaces or elsewhere, receptor proteins,glycoproteins, and the like, which are capable of interacting or bindingwith the analog. As an example, a deoxo-proline tamandarin or didemninanalog having the structure of formula I or XXI and attached to asupport can, by virtue of its interaction with a particular cellularreceptor, be used to identify or physically isolate cells of aparticular type (e.g. tumor cells) which are characterized by thepresence of the particular receptor.

Methods of Making Compounds Described Herein

Methods of making tamandarin and didemnin analogs have been described(e.g., Harris et al., 1987, Tetrahedron Lett. 28:2837-2840; Harris etal., 1988, Tetrahedron 44:3489-3500; Ewing et al., 1986, Tetrahedron42:5863-5868; Ewing, W. R., 1988, Ph.D. Dissertation, University ofPennsylvania, Philadelphia Pa.; Ewing et al., 1989, Tetrahedron Lett.30:3757-3760; Li et al., 1990, J. Am. Chem. Soc. 112:7659-7672; Mayer etal., 1994, J. Org. Chem. 59:5192-5205; Mayer et al., 1994, Tetrahedron:Asymmetry 5:519-522; Xiao et al., 1997, Tetrahedron: Asymmetry 9:47-53;Pfizenmayer et al., 1998, 8:3653-3656; U.S. patent application Ser. No.09/545,848, filed Apr. 7, 2000). The contents of each of thesereferences and patent application are incorporated herein by reference.The precise method used to make a tamandarin or didemnin macrocycle oranalog is not critical.

What represents novel subject matter in this disclosure is incorporationof a deoxo-proline residue or a dehydro-proline residue in the sidechain of a tamandarin or didemnin analog. The deoxo-proline residue ordehydro-proline residue can be used in place of a proline residue in anyknown tamandarin or didemnin analog. Particularly contemplatedtamandarin and didemnin analogs are those in which the deoxo-prolineresidue or dehydro-proline residue is linked to the macrocycle by way ofa leucine residue having a methylated amine moiety (i.e., an—(N-methyl)leucine-(deoxo or dehydro)-proline-containing tamandarin ordidemnin analog). Of course, the (deoxo or dehydro)-proline residue canbe further substituted, for example by lactate, by pyruvate, bylactate-fluorophore, by lactate-glutamine-pyroglutamate, bylactate-glutamine-cyclopentanoate, by -alanine-leucine-pyroglutamate, orby —(N-methyl-alanine)-leucine-pyroglutamate. One or more of the (deoxoor dehydro)-proline, lactate, glutamine, pyroglutamate, cyclopentanoate,and alanine residues is preferably the (S) enantiomer.

Incorporation of a deoxo-proline residue can be achieved by any methodknown in the art. Examples of methods of incorporating a deoxo-prolineresidue are included in this disclosure in Example 1 and in FIGS. 3-5.

In one embodiment, the method of incorporating a deoxo-proline residuecomprises protecting the hydroxyl and amine moieties of leucine,methylating the leucine amine moiety, and de-protecting the leucineamine moiety. The amine group of proline is protected, and the esterfunction of the proline is reduced to an aldehyde (e.g., using a strongbase such as LiBH₄ coupled with oxidation with an oxidizing agent suchas pyridine-SO₃). Reductive amination (e.g., in the presence of anon-aqueous solvent, a strong base, and a carboxylic acid catalyst;e.g., in the presence of Na(AcO)₃BH, AcOH, and CH₂Cl₂) can be used tocouple the hydroxyl-protected leucine with the amine protected proline(e.g., to form compound 43 in FIG. 5, in one embodiment). The reductiveamination can, for example, be performed as described by Abdel-Magid andco-workers (e.g., Abdel-Magid et al., 1990, Tetrahedron Lett.31:5595-5598; Abdel-Magid et al., 1990, Synlett. 537-539).

The resulting leucine-deoxo-proline dipeptide can be further substitutedwith other moieties (e.g., with -lactate, -pyruvate,-lactate-fluorophore, -lactate-glutamine-pyroglutamate,-lactate-glutamine-cyclopentanoate, -alanine-leucine-pyroglutamate, or—(N-methyl-alanine)-leucine-pyroglutamate), with (preferably) or withoutfirst removing the protecting groups. The leucine-deoxo-prolinedipeptide (optionally further substituted) can be attached to atamandarin or didemnin macrocycle in the position identified as R¹ informulas I and XXI.

Incorporation of a dehydro-proline residue can be achieved by any methodknown in the art. Examples of methods of incorporating a dehydro-prolineresidue are included in this disclosure in Example 2 and in FIG. 6.

In one embodiment, the method of incorporating a dehydro-proline residuecomprises protecting the carboxyl and amino groups of4-hydroxyprolinate, mesylating the 4-hydroxyl moiety, displacing themesylate moiety with an aryl-selenyl moiety, oxidatively eliminating thearyl-selenyl moiety, and de-protecting the carboxyl moiety. Theresulting dehydro-proline moiety can be coupled with one or moreadditional amino acid residues or organic acids (e.g., those identifiedherein, removing the amino-protecting group if necessary or desired) andcoupled with a tamandarin or didemnin macrocycle made or obtained byconventional means.

Pharmaceutical Compositions

The invention encompasses pharmaceutical compositions comprising atleast one of the deoxo-proline-containing tamandarin and didemninanalogs and dehydro-proline-containing tamandarin and didemnin analogsdescribed herein. Such compositions can comprise the analog and apharmaceutically acceptable carrier. By way of example, a pharmaceuticalcomposition can comprise a pharmaceutically acceptable carrier and atamandarin or didemnin analog having the structure of any of formulas I,II, and (a)-(d) as an active agent. As a further example, apharmaceutical composition can comprise a pharmaceutically-acceptablecarrier and one or more of the compounds depicted in the figures in thisdisclosure.

Such pharmaceutical compositions can be used, for example, in themethods described herein for and for inhibiting one or more of proteinsynthesis, cell cycle progression, tumorigenesis, growth, andproliferation in a cell. In addition, such compositions can be used inthe methods described herein for enhancing apoptosis in a cell.

Pharmaceutical compositions that are useful in the methods of theinvention can be administered systemically in oral solid formulations,ophthalmic, suppository, aerosol, topical or other similar formulations.In addition to the active agent, such pharmaceutical compositions cancontain pharmaceutically-acceptable carriers and other ingredients knownto enhance and facilitate drug administration. Other possibleformulations, such as nanoparticles, liposomes, resealed erythrocytes,and immunologically based systems can also be used to administer theactive agent according to the methods of the invention.

The invention encompasses pharmaceutical compositions which consist ofthe active agent, in a form suitable for administration to a subject, orthe pharmaceutical composition can comprise the active agent and one ormore pharmaceutically acceptable carriers, one or more additionalingredients, or some combination of these. The active agent can bepresent in the pharmaceutical composition in the form of aphysiologically acceptable ester or salt, such as in combination with aphysiologically acceptable cation or anion, as is well known in the art.

The formulations of the pharmaceutical compositions described herein canbe prepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active agent into association with a carrier or one or moreother accessory ingredients, and then, if necessary or desirable,shaping or packaging the product into a desired single- or multi-doseunit.

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for ethical administration to humans, it will be understood bythe skilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and perform such modification with merely ordinary, if any,experimentation. Subjects to which administration of the pharmaceuticalcompositions of the invention is contemplated include, but are notlimited to, humans and other primates, and mammals includingcommercially relevant mammals such as cattle, pigs, horses, sheep, cats,and dogs.

Pharmaceutical compositions that are useful in the methods of theinvention can be prepared, packaged, or sold in formulations suitablefor oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal,buccal, ophthalmic, or another route of administration. Othercontemplated formulations include projected nanoparticles, liposomalpreparations, resealed erythrocytes containing the active agent, andimmunologically-based formulations.

A pharmaceutical composition of the invention can be prepared, packaged,or sold in bulk, as a single unit dose, or as a plurality of single unitdoses. As used herein, a “unit dose” is discrete amount of thepharmaceutical composition comprising a predetermined amount of theactive agent. The amount of the active agent is generally equal to thedosage of the active agent which would be administered to a subject or aconvenient fraction of such a dosage such as, for example, one-half orone-third of such a dosage.

In addition to the active agent, a pharmaceutical composition of theinvention can further comprise one or more additional pharmaceuticallyactive agents such as, other tumor therapy agents, other anti-infectiveagents, and the like.

Controlled- or sustained-release formulations of a pharmaceuticalcomposition of the invention can be made using conventional technology.

A formulation of a pharmaceutical composition of the invention suitablefor oral administration can be prepared, packaged, or sold in the formof a discrete solid dose unit including, but not limited to, a tablet, ahard or soft capsule, a cachet, a troche, or a lozenge, each containinga predetermined amount of the active agent. Other formulations suitablefor oral administration include, but are not limited to, a powdered orgranular formulation, an aqueous or oily suspension, an aqueous or oilysolution, or an emulsion.

As used herein, an “oily” liquid is one which comprises acarbon-containing liquid molecule and which exhibits a less polarcharacter than water.

A tablet comprising the active agent may, for example, be made bycompressing or molding the active agent, optionally with one or moreadditional ingredients. Compressed tablets can be prepared bycompressing, in a suitable device, the active agent in a free-flowingform such as a powder or granular preparation, optionally mixed with oneor more of a binder, a lubricant, an excipient, a surface active agent,and a dispersing agent. Molded tablets can be made by molding, in asuitable device, a mixture of the active agent, a pharmaceuticallyacceptable carrier, and at least sufficient liquid to moisten themixture. Pharmaceutically acceptable excipients used in the manufactureof tablets include, but are not limited to, inert diluents, granulatingand disintegrating agents, binding agents, and lubricating agents. Knowndispersing agents include, but are not limited to, potato starch andsodium starch glycollate. Known surface active agents include, but arenot limited to, sodium lauryl sulfate. Known diluents include, but arenot limited to, calcium carbonate, sodium carbonate, lactose,microcrystalline cellulose, calcium phosphate, calcium hydrogenphosphate, and sodium phosphate. Known granulating and disintegratingagents include, but are not limited to, corn starch and alginate. Knownbinding agents include, but are not limited to, gelatin, acacia,pre-gelatinized maize starch, polyvinylpyrrolidone, and hydroxypropylmethylcellulose. Known lubricating agents include, but are not limitedto, magnesium stearate, stearate, silica, and talc.

Tablets can be non-coated or they can be coated using known methods toachieve delayed disintegration in the gastrointestinal tract of asubject, thereby providing sustained release and absorption of theactive agent. By way of example, a material such as glycerylmonostearate or glyceryl distearate can be used to coat tablets. Furtherby way of example, tablets can be coated using methods described in U.S.Pat. Nos. 4,256,108; 4,160,452; and 4,265,874 to formosmotically-controlled release tablets. Tablets can further comprise asweetening agent, a flavoring agent, a coloring agent, a preservative,or some combination of these in order to provide pharmaceuticallyelegant and palatable preparation.

Hard capsules comprising the active agent can be made using aphysiologically degradable composition, such as gelatin. Such hardcapsules comprise the active agent, and can further comprise additionalingredients including, for example, an inert solid diluent such ascalcium carbonate, calcium phosphate, or kaolin.

Soft gelatin capsules comprising the active agent can be made using aphysiologically degradable composition, such as gelatin. Such softcapsules comprise the active agent, which can be mixed with water or anoil medium such as peanut oil, liquid paraffin, or olive oil.

Liquid formulations of a pharmaceutical composition of the inventionwhich are suitable for oral administration can be prepared, packaged,and sold either in liquid form or in the form of a dry product intendedfor reconstitution with water or another suitable vehicle prior to use.

Liquid suspensions can be prepared using conventional methods to achievesuspension of the active agent in an aqueous or oily vehicle. Aqueousvehicles include, for example, water and isotonic saline. Oily vehiclesinclude, for example, almond oil, oily esters, ethyl alcohol, vegetableoils such as arachis, olive, sesame, or coconut oil, fractionatedvegetable oils, and mineral oils such as liquid paraffin. Liquidsuspensions can further comprise one or more additional ingredientsincluding, but not limited to, suspending agents, dispersing or wettingagents, emulsifying agents, demulcents, preservatives, buffers, salts,flavorings, coloring agents, and sweetening agents. Oily suspensions canfurther comprise a thickening agent. Known suspending agents include,but are not limited to, sorbitol syrup, hydrogenated edible fats, sodiumalginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, andcellulose derivatives such as sodium carboxymethylcellulose,methylcellulose, hydroxypropylmethylcellulose. Known dispersing orwetting agents include, but are not limited to, naturally-occurringphosphatides such as lecithin, condensation products of an alkyleneoxide with a fatty acid, with a long chain aliphatic alcohol, with apartial ester derived from a fatty acid and a hexitol, or with a partialester derived from a fatty acid and a hexitol anhydride (e.g.polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylenesorbitol monooleate, and polyoxyethylene sorbitan mono-oleate,respectively). Known emulsifying agents include, but are not limited to,lecithin and acacia. Known preservatives include, but are not limitedto, methyl, ethyl, or n-propyl-para-hydroxybenzoates, ascorbate, andsorbate. Known sweetening agents include, for example, glycerol,propylene glycol, sorbitol, sucrose, and saccharin. Known thickeningagents for oily suspensions include, for example, beeswax, hardparaffin, and cetyl alcohol.

Liquid solutions of the active agent in aqueous or oily solvents can beprepared in substantially the same manner as liquid suspensions, theprimary difference being that the active agent is dissolved, rather thansuspended in the solvent. Liquid solutions of the pharmaceuticalcomposition of the invention can comprise each of the componentsdescribed with regard to liquid suspensions, it being understood thatsuspending agents will not necessarily aid dissolution of the activeagent in the solvent. Aqueous solvents include, for example, water andisotonic saline. Oily solvents include, for example, almond oil, oilyesters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, orcoconut oil, fractionated vegetable oils, and mineral oils such asliquid paraffin.

Powdered and granular formulations of a pharmaceutical preparation ofthe invention can be prepared using known methods. Such formulations canbe administered directly to a subject, used, for example, to formtablets, to fill capsules, or to prepare an aqueous or oily suspensionor solution by addition of an aqueous or oily vehicle thereto. Each ofthese formulations can further comprise one or more of dispersing orwetting agent, a suspending agent, and a preservative. Additionalexcipients, such as fillers and sweetening, flavoring, or coloringagents, can also be included in these formulations.

A pharmaceutical composition of the invention can also be prepared,packaged, or sold in the form of oil-in-water emulsion or a water-in-oilemulsion. The oily phase can be a vegetable oil such as olive or arachisoil, a mineral oil such as liquid paraffin, or a combination of these.Such compositions can further comprise one or more emulsifying agentssuch as naturally occurring gums such as gum acacia or gum tragacanth,naturally-occurring phosphatides such as soybean or lecithinphosphatide, esters or partial esters derived from combinations of fattyacids and hexitol anhydrides such as sorbitan monooleate, andcondensation products of such partial esters with ethylene oxide such aspolyoxyethylene sorbitan monooleate. These emulsions can also containadditional ingredients including, for example, sweetening or flavoringagents.

A pharmaceutical composition of the invention can be prepared, packaged,or sold in a formulation suitable for rectal administration. Such acomposition can be in the form of, for example, a suppository, aretention enema preparation, and a solution for rectal or colonicirrigation.

Suppository formulations can be made by combining the active agent witha non-irritating pharmaceutically acceptable excipient which is solid atordinary room temperature (i.e. about 20° C.) and which is liquid at therectal temperature of the subject (i.e. about 37° C. in a healthyhuman). Suitable pharmaceutically acceptable excipients include, but arenot limited to, cocoa butter, polyethylene glycols, and variousglycerides. Suppository formulations can further comprise variousadditional ingredients including, but not limited to, antioxidants andpreservatives.

Retention enema preparations or solutions for rectal or colonicirrigation can be made by combining the active agent with apharmaceutically acceptable liquid carrier. As is well known in the art,enema preparations can be administered using, and can be packagedwithin, a delivery device adapted to the rectal anatomy of the subject.Enema preparations can further comprise various additional ingredientsincluding, but not limited to, antioxidants and preservatives.

A pharmaceutical composition of the invention can be prepared, packaged,or sold in a formulation suitable for vaginal administration. Such acomposition can be in the form of, for example, a suppository, animpregnated or coated vaginally-insertable material such as a tampon, adouche preparation, or a solution for vaginal irrigation.

Methods for impregnating or coating a material with a chemicalcomposition are known in the art, and include, but are not limited tomethods of depositing or binding a chemical composition onto a surface,methods of incorporating a chemical composition into the structure of amaterial during the synthesis of the material (i.e. such as with aphysiologically degradable material), and methods of absorbing anaqueous or oily solution or suspension into an absorbent material, withor without subsequent drying.

Douche preparations or solutions for vaginal irrigation can be made bycombining the active agent with a pharmaceutically acceptable liquidcarrier. As is well known in the art, douche preparations can beadministered using, and can be packaged within, a delivery deviceadapted to the vaginal anatomy of the subject. Douche preparations canfurther comprise various additional ingredients including, but notlimited to, antioxidants, antibiotics, anti-fungal agents, andpreservatives.

Formulations of a pharmaceutical composition suitable for parenteraladministration can comprise the active agent combined with apharmaceutically acceptable carrier, such as sterile water or sterileisotonic saline. Such formulations can be prepared, packaged, or sold ina form suitable for bolus administration or for continuousadministration. Injectable formulations can be prepared, packaged, orsold in unit dosage form, such as in ampules or in multi-dose containerscontaining a preservative. Formulations for parenteral administrationinclude, but are not limited to, suspensions, solutions, emulsions inoily or aqueous vehicles, pastes, and implantable sustained-release orbiodegradable formulations. Such formulations can further comprise oneor more additional ingredients including, but not limited to,suspending, stabilizing, or dispersing agents. In one embodiment of aformulation for parenteral administration, the active agent is providedin dry (i.e. powder or granular) form for reconstitution with a suitablevehicle (e.g. sterile pyrogen-free water) prior to parenteraladministration of the reconstituted composition.

The pharmaceutical compositions can be prepared, packaged, or sold inthe form of a sterile injectable aqueous or oily suspension or solution.This suspension or solution can be formulated according to the knownart, and can comprise, in addition to the active agent, additionalingredients such as the dispersing agents, wetting agents, or suspendingagents described herein. Such sterile injectable formulations can beprepared using a non-toxic parenterally-acceptable diluent or solvent,such as water or 1,3-butane diol, for example. Other acceptable diluentsand solvents include, but are not limited to, Ringer's solution,isotonic sodium chloride solution, and fixed oils such as syntheticmono- or di-glycerides. Other parentally-administrable formulationswhich are useful include those which comprise the active agent inmicrocrystalline form, in a liposomal preparation, or as a component ofa biodegradable polymer systems. Compositions for sustained release orimplantation can comprise pharmaceutically acceptable polymeric orhydrophobic materials such as an emulsion, an ion exchange resin, asparingly soluble polymer, or a sparingly soluble salt.

Formulations suitable for topical administration include, but are notlimited to, liquid or semi-liquid preparations such as liniments,lotions, oil-in-water or water-in-oil emulsions such as creams,ointments or pastes, and solutions or suspensions.Topically-administrable formulations may, for example, comprise fromabout 1% to about 10% (w/w) active agent, although the concentration ofthe active agent can be as high as the solubility limit of the activeagent in the solvent. Formulations for topical administration canfurther comprise one or more of the additional ingredients describedherein.

A pharmaceutical composition of the invention can be prepared, packaged,or sold in a formulation suitable for pulmonary administration via thebuccal cavity. Such a formulation can comprise dry particles whichcomprise the active agent and which have a diameter in the range fromabout 0.5 to about 7 nanometers, and preferably from about 1 to about 6nanometers. Such compositions are conveniently in the form of drypowders for administration using a device comprising a dry powderreservoir to which a stream of propellant can be directed to dispersethe powder or using a self-propelling solvent/powder-dispensingcontainer such as a device comprising the active agent dissolved orsuspended in a low-boiling propellant in a sealed container. Preferably,such powders comprise particles wherein at least 98% of the particles byweight have a diameter greater than 0.5 nanometers and at least 95% ofthe particles by number have a diameter less than 7 nanometers. Morepreferably, at least 95% of the particles by weight have a diametergreater than 1 nanometer and at least 90% of the particles by numberhave a diameter less than 6 nanometers. Dry powder compositionspreferably include a solid fine powder diluent such as sugar and areconveniently provided in a unit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generally thepropellant can constitute 50 to 99.9% (w/w) of the composition, and theactive agent can constitute 0.1 to 20% (w/w) of the composition. Thepropellant can further comprise additional ingredients such as a liquidnon-ionic or solid anionic surfactant or a solid diluent (preferablyhaving a particle size of the same order as particles comprising theactive agent).

Pharmaceutical compositions of the invention formulated for pulmonarydelivery can also provide the active agent in the form of droplets of asolution or suspension. Such formulations can be prepared, packaged, orsold as aqueous or dilute alcoholic solutions or suspensions, optionallysterile, comprising the active agent, and can conveniently beadministered using any nebulization or atomization device. Suchformulations can further comprise one or more additional ingredientsincluding, but not limited to, a flavoring agent such as saccharinsodium, a volatile oil, a buffering agent, a surface active agent, or apreservative such as methylhydroxybenzoate. The droplets provided bythis route of administration preferably have an average diameter in therange from about 0.1 to about 200 nanometers.

The formulations described herein as being useful for pulmonary deliveryare also useful for intranasal delivery of a pharmaceutical compositionof the invention.

Another formulation suitable for intranasal administration is a coarsepowder comprising the active agent and having an average particle fromabout 0.2 to 500 micrometers. Such a formulation is administered in themanner in which snuff is taken i.e. by rapid inhalation through thenasal passage from a container of the powder held close to the nares.

Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) ofthe active agent, and can further comprise one or more of the additionalingredients described herein.

A pharmaceutical composition of the invention can be prepared, packaged,or sold in a formulation suitable for buccal administration. Suchformulations may, for example, be in the form of tablets or lozengesmade using conventional methods, and may, for example, 0.1 to 20% (w/w)active agent, the balance comprising an orally dissolvable or degradablecomposition and, optionally, one or more of the additional ingredientsdescribed herein. Alternately, formulations suitable for buccaladministration can comprise a powder or an aerosolized or atomizedsolution or suspension comprising the active agent. Such powdered,aerosolized, or aerosolized formulations, when dispersed, preferablyhave an average particle or droplet size in the range from about 0.1 toabout 200 nanometers, and can further comprise one or more of theadditional ingredients described herein.

A pharmaceutical composition of the invention can be prepared, packaged,or sold in a formulation suitable for ophthalmic administration. Suchformulations may, for example, be in the form of eye drops including,for example, a 0.1-1.0% (w/w) solution or suspension of the active agentin an aqueous or oily liquid carrier. Such drops can further comprisebuffering agents, salts, or one or more other of the additionalingredients described herein. Other ophthalmalmically-administrableformulations which are useful include those which comprise the activeagent in microcrystalline form or in a liposomal preparation.

As used herein, “additional ingredients” include, but are not limitedto, one or more of the following: excipients; surface active agents;dispersing agents; inert diluents; granulating and disintegratingagents; binding agents; lubricating agents; sweetening agents; flavoringagents; coloring agents; preservatives; physiologically degradablecompositions such as gelatin; aqueous vehicles and solvents; oilyvehicles and solvents; suspending agents; dispersing or wetting agents;emulsifying agents, demulcents; buffers; salts; thickening agents;fillers; emulsifying agents; antioxidants; antibiotics; anti-fungalagents; stabilizing agents; and pharmaceutically acceptable polymeric orhydrophobic materials. Other “additional ingredients” which can beincluded in the pharmaceutical compositions of the invention are knownin the art and described, for example in Genaro, ed., 1985, Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., which isincorporated herein by reference.

The relative amounts of the active agent, the pharmaceuticallyacceptable carrier, and any additional ingredients in a pharmaceuticalcomposition of the invention will vary, depending upon the identity,size, and the type and severity of condition of the subject treated andfurther depending upon the route by which the composition is to beadministered. By way of example, the composition can comprise between 0.1% and 100% (w/w) active agent.

Typically dosages of the active agent which can be administered to ananimal, preferably a human, range in amount from 1 microgram to about100 grams per kilogram of body weight of the animal. While the precisedosage administered will vary depending upon any number of factors,including but not limited to, the type of animal and type of diseasestate being treated, the age of the animal and the route ofadministration. Preferably, the dosage of the active agent will varyfrom about 1 milligram to about 10 g per kilogram of body weight of theanimal. More preferably, the dosage will vary from about 10 milligram toabout 1 gram per kilogram of body weight of the animal. Alternatively,the dosage can be determined in units of square meters of the bodysurface of an animal (i.e. milligrams or kilograms per square meter,mg/m² or kg/m²). Preferably, this dosage will vary from about 0.1milligram to about 5 grams per square meter of body surface of theanimal. More preferably, the dosage will vary from about 1 milligram toabout 1 gram per square meter of body surface of the animal.

The active agent can be administered to an animal as frequently asseveral times daily, or it can be administered less frequently, such asonce a day, once a week, once every two weeks, once a month, or evenlees frequently, such as once every several months or even once a yearor less. The frequency of the dose is determinable by the skilledartisan and depends upon various factors including, but not limited to,the type and severity of the disease being treated, the type and age ofthe animal, etc.

The invention is now described with reference to the following Examples.These Examples are provided for the purpose of illustration only and theinvention is not limited to these Examples, but rather encompasses allvariations which are evident as a result of the teaching providedherein.

EXAMPLES

Unless otherwise stated, all reactions were conducted in the presence ofan inert atmosphere (e.g. argon or nitrogen). All solvents were reagentgrade (e.g. distilled solvents, chromatography solvents, and reactionwork-up solvents) or HPLC grade (i.e. reaction solvents). Anhydrousdiethyl ether and tetrahydrofuran (THF) were distilled from sodium andbenzophenone. The boiling point range of the hexane used was 38-55° C.Methylene chloride (CH₂Cl₂), benzene, toluene, and N,N-dimethylfornamide (DMF) were distilled from calcium hydride (CaH₂). Organicacids and bases were reagent grade. Triethylamine (Et₃N),diisopropylethylamine (DIPEA), morpholine, and N-methylmorpholine (NMM)were distilled from calcium hydride (CaH₂). All other reagents,including dimethylaminophenol and diethyl 1,3-acetonedicarboxylate, werethe highest purity commercial available. Analytical thin-layerchromatography (TLC) was performed using EM Separations Tech./Mercksilica gel (60-F254) plates (0.25 millimeter) pre-coated with afluorescent indicator. Visualization was effected using ultravioletlight (254 nanometers), phosphomolybdic acid (7% w/v) in 95% ethanol.Melting points (mp) were determined using a Thomas-Hoover capillarymelting point apparatus and are reported without correction. Proton andcarbon magnetic resonance spectra (¹H— and ¹³C-NMR, respectively) wererecorded on a Bruker AM-500 (500 MHz) Fourier transform spectrometer,and chemical shifts were expressed in parts per million (ppm) relativeto CHCl₃ as an internal reference (7.24 ppm for ¹H and 77.0 for C).Multiplicities are designated as singlet (s), doublet (d), doublet ofdoublets (dd), doublet of triplets (dt), triplet (t), quartet (q)multiplet (m), and broad singlet (s). Infrared spectra (IR) wereobtained using a Perkin-Elmer Model 1600 FT-IR spectrophotometer.Absorptions are reported in wave number (cm⁻¹). Optical rotations (indegrees) were measured using a Perkin-Elmer Model 341 polarimeter. Highresolution mass spectra (HRMS) were obtained using either a VG 70-70HS,or a Micromass AutoSpect. Elemental Analyses were performed using aPerkin-Elmer 2400 Series II CHNS/O Analyzer. Flash column chromatographywas performed using Merck silica gel 60 (240-400 mesh) using the solventsystems indicated for individual experiments.

Example 1 Synthesis of a Deoxo-Proline Side Chain Moiety and Coupling toa Didemnin Macrocycle

An amino methylene single bond was used to replace the amide bondbetween D-leucine and L-proline in the side chain of didemnin B.Synthetically, the amino methylene bond was prepared by reductiveamination, as described (Abdel-Magid et al., 1990, Tetrahedron Lett.31:5595-5598; Abdel-Magid et al., 1990, Synlett. 537-539).

In a first synthetic method, exhaustive methylation of Cbz-D-leucine wasperformed using dimethylsulfate, as illustrated in FIG. 3. The Cbz groupwas removed using hydrogenolysis to yield dimethylated D-leucine freeamine (Compound 40). The amine was used in reductive amination withoutpurification. Commercially available L-proline was first esterifiedusing SOCl₂ in MeOH, and its amino group was subsequently protectedusing Boc₂O. After purification, the ester function was reduced to analdehyde in two steps. NaBH₄ was combined with LiCl to generate LiBH₄ insitu, and this compound was used to reduce the ester to thecorresponding alcohol. Oxidation with SO₃.pyridine reagent gave thealdehyde, designated compound 42, in good yield. Reduction of the esterto the aldehyde in one step using DIBAL could be performed, but dependedon the freshness of the reducing reagent and was not very reproducible.Reductive amination between the aldehyde designated compound 40 and thefree amine (compound 42) yielded compound 43. The Boc group of compound43 was removed using TFA/CH₂Cl₂. The resulting free amine was coupledwith pyruvic acid using BOP to yield the protected Ψ(CH₂NH) side chaindesignated compound 44.

The next step was hydrolysis of the methyl ester of compound 44. Butthis step was proved to be nontrivial. Perhaps owing to sterichindrance, the methyl ester was difficult to cleave using 2 equivalentsof LiOH.H₂O in THF/H₂O. When the amount of LiOH.H₂O was increased to 10equivalents, the methyl ester appeared to hydrolyze, since the massspectrum of the reaction mixture did show the acid peak. But the acidwas so hydrophilic and buried inside the excess inorganic salts, that itcould not be extracted by any organic solvent. We also tried toprecipitate the acid using ether but only precipitated the inorganicsalts. On account of this purification problem, we decided not to usethe methyl ester to protect the acid. We needed a protective group whichcould survive all the synthetic steps but did not require aqueousconditions for its removal. The benzyl group was found to serve thispurpose well.

The synthetic procedure was modified as indicated in FIG. 4. In order tobe compatible with the benzyl ester, the leucine amino group wasprotected by Boc instead of Cbz. Selective methylation of the aminogroup gave the acid designated compound 46. The free acid was benzylatedto yield the desired benzyl ester, compound 47. The Boc protecting groupwas removed using TFA. The resulting amine (compound 48) was condensedwith protected prolinal by reductive amination. Removal of the Bocprotecting group and subsequent coupling with pyruvic acid yieldedcompound 50. Hydrogenolysis was used to remove the benzyl group andyielded the desired Ψ(CH₂NH) side chain, in the form of the free acid(compound 51). Coupling of the free acid side chain with a didenminmacrocycle gave the desired deoxo-proline didemnin analog designatedcompound 12.

A second deoxo-proline didemnin analog (designated compound 72 a) wassynthesized using a similar synthetic strategy, as illustrated in FIG. 5and described in detail as follows.

Benzyl N-Boc D-leucinate (Compound 46)

A solution of N-Boc-D-leucine (1.0 gram, 4.1 millimoles) in 20milliliters of DMF was cooled to 0° C. Finely powdered Li₂CO₃ (1.5grams, 20.5 millimoles) was added, followed by the addition of benzylbromide (2.43 milliliters, 20.5 millimoles). The reaction mixture wasstirred for 6 hours and monitored by TLC. When the reaction wascomplete, the reaction mixture was diluted with H₂O and extracted withEtOAc three times. The EtOAc extracts were combined and washed withbrine. DMF was removed in vacuo. The crude mixture was purified withcolumn chromatography eluting with 20% acetone/hexane to afford 46 in78% yield. The following analytical data were obtained for compound 46:R_(f) 0.60 (40% acetone/hexane); [α]_(D) ²⁵+16 (c=1.0, CHCl₃); ¹H NMR(500 MHz, CDC₃) δ H_(a)) 0.92 (m, 6H), H_(b)) 1.48 (s, 9H), H_(c))1.50-1.59 (m, 1H), H_(d)) 1.60-1.69 (dd, 2H), H_(e)) 4.36 (m, 1H),H_(f)) 4.90 (d, 1H), H_(g)) 5.11-5.20 (m, 2H), H_(h)) 7.33 (m, 5H); ¹³CNMR (125 MHz, CDCl₃) δC_(a)) 21.8, C_(a)′) 22.7, C_(c)) 24.7, C_(b))28.2, C_(d)) 41.6, C_(e)) 52.1, C_(g)) 66.7, Ci) 79.6, C_(h)) 128.0,128.2, 128.4, C_(l)) 135.5, C_(j)) 155.3, C_(k)) 173.2; IR (neat) 3367(br), 2958 (s), 1732 (s), 1715 (s), 1500 (s), 1455 (m), 1366 (w), 1120(m) cm⁻¹; HRMS m/z calculated for C₁₁H₂₃N₄O₂ (M+H) 322.2017, found322.2018.

N-Boc N-Methyl benzyl D-leucinate (Compound 47)

A solution of compound 46 (1.2 grams, 3.74 millimoles) in 200milliliters of THF was cooled to 0° C. NaHMDS (1 molar in THF; 5.6milliliters, 5.6 millimoles) was added, followed by the addition ofmethyl iodide (1.0 milliliter, 18.7 millimoles). The reaction mixturewas stirred overnight and monitored by TLC. When the reaction wascomplete, the reaction mixture was diluted with ether. The organic layerwas washed with 5% HCl, 5% NaHCO₃, and brine. The resulting solution wasdried over Na₂SO₄ and concentrated. The crude mixture was purified bycolumn chromatography, eluting with 20% acetone/hexane to afford 47 in71% yield. The following analytical data were obtained for compound 47:R_(f) 0.55 (30% Acetone/Hexane); [α]_(D) ²⁵+20.4 (c=1.2, CHCl₃); ¹H NMR(500 MHz, CDCl₃) δ H_(a)) 0.85-0.90 (m, 6H), H_(c)) & H_(d)) 1.35-1.65(m, 3H), H_(b)) 1.45 (s, 9H), H_(e)) 2.75 (d, 3H), H^(f)) 4.53-4.58 &4.81-4.88 (rm, 1H), H_(g)) 5.10 (s, 2H), H_(h)) 7.15-7.28 (m, 5H); ¹³CNMR (125 MHz, CDCl₃) δ C_(a)) 22.0 and 23.5, C_(c)) 25.8, C_(b)) 29.0,C_(d)) 31.0, C_(e)) 38.0, C_(f)) 57.0, C_(g)) 66.1, C_(i)) 80.2, Ch)128.0, 128.2, 128.4, Cl) 136.5, C_(j)) 156.3, C_(k)) 173.2; IR (neat)2958.3 (m), 1742.7 (s), 1696.8 (s), 1455.6 (s), 1390.7 (s), 1366.6 (s),1323.5 (s), 1151.3 (s) cm⁻¹; HRMS m/z calculated for C₁₁H₂₃N₄O₂ (M+H)336.2174, found 336.2178.

Benzyl N-Methyl D-leucinate hydrochloride salt (Compound 48)

Compound 47 (0.1 gram) was dissolved in HCl.dioxane (5 milliliters) andstirred at room temperature. When the reaction was completed, thesolvent was removed in vacuo. Toluene was added twice and concentrated.The residue was dried under reduced pressure overnight to afford thedesired HCl salt (compound 48) in 98%. The following analytical datawere obtained for compound 48: R_(f): baseline (10% MeOH/CH₂Cl₂);[α]_(D) ²⁵+48.5 (c=0.2, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ H_(a)) 0.91(d, 6H), H_(b)) 1.75 (m, 1H) H_(c)) 1.90-1.95 (dd, 2H), H_(d)) 2.71 (s,3H), H_(e)) 3.83 (t, 1H), H_(f)) 5.20-5.30 (rm, 2H), H_(g)) 7.35-7.40(m, 5H), H_(h)) 9.85 & 10.15 (br, 2H); ¹³C NMR (125 MHz, CDCl₃) δ C_(a))21.8, and 23.4, Cc) 25.8, C_(b)) 31.9, C_(d)) 38.2, C_(e)) 60.1, C_(f))68.8, C_(g)) 128.4, 128.6, 128.8, C_(j)) 134.6, C_(i)) 168.2; IR (neat)2958.3 (m), 1742 (s), 1696 (s), 1455 (s), 1390 (s), 1366 (s), 1323 (s),1151 (s) cm⁻¹; HRMS m/z calculated for C₁₁ H₂₃N₄O₂ (M+H) 336.2174, found336.2178.

Methyl N-Boc L-Prolinate (Compound 41)

Commercially available N-Boc-L-proline (5.0 grams, 23.2 millimoles) wasdissolved in acetone (200 milliliters). K₂CO₃ (3.8 grams, 27.84millimoles) was added at 0° C., followed by the addition of MeI (2.9milliliters, 46.4 millimoles). The reaction mixture was stirredovernight. The reaction was quenched with 5% NaHCO₃ solution andextracted with ether. The combined ether layers were washed with 5% HCland brine, and concentrated to afford the crude product (compound 41) in91% yield. This compound was purified by column chromatography elutingwith 20% acetone/hexane. The following analytical data were obtained forcompound 41: R_(f) 0.4 (30% acetone/hexane); [α]_(D) ²⁵−61.2 (c=0.8,CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ H_(a)) 1.45 (s, 9H), H_(b)) 1.98 (dd,2H) H_(c)) 1.88 & 2.22 (m, rm, 2H), H_(d)) 3.42-3.58 (m, 2H), H_(e))3.74 (s, 3H), H_(f)) 4.24 & 4.35 (rm, 1H); ¹³C NMR (125 MHz, CDCl₃) δC_(b)) 24.2, and 24.4, Ca) 28.3, C_(c)) 31.6 and 31.8, C_(d)) 47.3,C_(f)) 52.1, C_(e)) 59.7, C_(g)) 80.1, C_(h)) 153.8, C_(i)) 174.0; IR(neat) 2975 (m), 1749 (s), 1700 (s), 1396 (s), 1365 (s), 1200 (s), 1161(s), 1151 (s); HRMS m/z calculated for C₁₁H₂₃N₄O₂ (M+H) 230.1391, found230.1389.

N-Boc L-Prolinol (Compound 41 a)

Compound 41 was dissolved in 200 milliliters THF/EtOH (1:1). LiCl (1.8grams, 32.7 millimoles) and NaBH₄ (1.2 grams, 32.7 millimoles) was addedin portions at 0° C. The reaction mixture was stirred overnight andmonitored by TLC; more LiCi and NaBH₄ were added during the operation.When the reaction was complete, the white solid was collected and washedwith ether. The solvent was removed using a rotary evaporator. Theresidue was neutralized to pH 4 and then extracted twice with EtOAc. TheEtOAc extracts were combined, washed with brine, dried, andconcentrated. The crude product was purified by column chromatographyeluting with 10% acetone/hexane to afford the desired alcohol (compound41 a) in 83% yield. The following analytical data were obtained forcompound 41 a: Rf 0.30 (30% Acetone/Hexane); [α]_(D) ²⁵−60.0 (c=0.8,CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ H_(a)) 1.48 (s, 9H), H_(b)) 1.60 &2.05 (rm, 2H) H_(c)) 1.83-1.98 (dd, 2H), H_(d)) 3.35 & 3.45 (rm, 2H),H_(e)) 3.61-3.70 (m, 2H), H_(f)) 4.03 (m, 1H); ¹³C NMR (125 MHz, CDCl₃)δ C_(c)) 23.8, Ca) 28.4, C_(b)) 28.9, C_(f)) 47.3, C_(e)) 59.9, C_(d))67.3, Cg) 80.0, C_(h)) 156.8; IR (neat) 3424 (br), 2973 (s), 2877 (s),1695 (s), 1670 (s), 1477 (s), 1406 (s), 1366.2 (s); HRMS m/z calculatedfor C₁₁H₂₃N₄O₂ (M+H) 202.1443, found 202.1449.

N-Boc L-prolinal (compound 42).

A solution of compound 41 a (2.0 grams, 9.9 millimoles) and Et₃N in 200milliliters of CH₂Cl₂ was cooled to −78° C. SO₃.pyridine complex (4.7grams, 29.7 millimoles) in DMSO (30 milliliters) was added to theprevious solution. The reaction mixture was warmed to room temperature,stirred overnight, and monitored by TLC. When the reaction was complete,the reaction mixture was diluted with ether. The organic layer waswashed with 5% HCl, 5% NaHCO₃, and brine. The resulting solution wasdried over Na₂SO₄ and concentrated. The crude mixture was purified witha short column to afford the desired aldehyde (compound 42) in 81%yield. The following analytical data were obtained for compound 42:R_(f) 0.55 (30% Acetone/Hexane); [α]_(D) ²⁵+20.4° (c=1.2, CHCl₃); ¹H NMR(500 MHz, CDCl₃) δ H_(a)) 1.45 (s, 9H), H_(b)) 1.75-1.90 (m, 2H), H_(c))1.90-2.15 (m, 2H), H_(d)) 3.20-3.50 (t, 2H), H_(e)) 3.90-4.20 (d, 1H),H_(f)) 9.72 (d, 1H); ¹³C NMR (125 MHz, CDCl₃) δ C_(b)) 24.0, Cc) 27.3,C_(a)) 28.9, C_(d)) 47.5, C_(e)) 65.5, Cg) 80.1, C_(h)) 154.2, C_(f))200.1.

N-Boc-pro y(NHCH₂) N-Methyl-Benzyl-D-Leucinate (compound 43).

Compound 42 (0.1 gram, 0.5 millimoles) was dissolved in CH₂Cl₂ (8milliliters), and free amine 48 (0.15 gram, 0.55 millimoles) was addedwith efficient stirring. At 0° C., AcOH (0.02 milliliters, 0.3millimoles) was added as a catalyst and stirred for 10 minutes beforeNaBH(OAc)3 (0.13 gram, 0.6 millimoles) was added to the reactionmixture. The reaction mixture was stirred at room temperature andmonitored by TLC. When the reaction was complete, the mixture wasdiluted with CH₂Cl₂. The excess reagent was quenched by dropwiseaddition of saturated NH₄Cl solution. The organic layer was washed with5% HCl, 5% NaHCO₃, and brine, dried, and concentrated. The crude productwas purified by flash chromatography, eluting with 20% EtOAc/petroleumether to afford the desired amine (compound 43). The followinganalytical data were obtained for compound 43: R_(f) 0.50 (30%Acetone/Hexane); [α]_(D) ²⁵ −44.0 (c=1.1, CHCl₃); ¹H NMR (500 MHz,CDCl₃) δ H_(a)) 0.92 (m, 6H), H_(b)) 1.45 (s, 9H), H_(c)), H_(d)) &H_(e)) 1.50-1.90 (m, 5H), H_(f)) & H_(g)) 2.3-2.45 (m, 5H), H_(h1))2.22-2.90 (m, 1H), H₂) & H_(i)) 3.25-3.4 (m, 3H), H_(j)) & H_(k))3.70-3.95 (m, 2H), H_(l)) 5.35 (m, 5H), H_(m)) 7.30 (m, 5H); ¹³C NMR(125 MHz, CDCl₃) δ C_(a)) 22.2, C_(d)) 23.4, C_(c)) 25.6, C_(b)) 28.4,C_(e)) 28.8, C_(g)) 36.6, C_(f)) 38.4, C_(h)) 46.2, C_(i)) 56.1, C_(j))58.5, C_(l)) 56.2, C_(k)) 66.0, C_(n)) 79.6, C_(m)) 128.2, C_(q)) 136.2,C_(o)) 154.1, C_(p)) 174.2; IR (neat) 2955 (s), 2868 (s), 1730 (s), 1693(s), 1455 (s), 1392 (s), 1364 (s), 1170 (s) cm⁻¹; HRMS m/z calculatedfor C₂₄H₃₈N₂O₄ (M+H) 419.2910, found 419.2897.

L-Pro Ψ(NHCH₂) N-Methyl-Benzyl-D-Leucinate Hydrochloride salt (compound44).

Compound 43 (0.1 gram) was dissolved in HCl-dioxane (5 milliliters), andthe solution was stirred at room temperature. When the reaction wascompleted, all solvent was removed in vacuo. Toluene was added twice andconcentrated. The residue was dried under reduced pressure overnight toafford the desired HCl salt (compound 44) in 98%. The followinganalytical data were obtained for compound 44: R_(f) baseline (60%EA/PE); [α]_(D) ²⁵ +15.3 (c=0.6, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δH_(a)) 0.91-1.10 (d, 6H), H_(b)), H_(c)) & H_(d)) 1.68-2.30 (m, 5H),H_(e)) 2.83 (dd, 2H), H_(f)) 3.12 (s, 3H), H_(g)) & H_(h)) 3.31-3.65 (m,4H), H_(i)) & H_(j)) 4.11-4.30 (m, 2H), H_(k)) 5.28 (s, 2H), H_(k)) 7.40(m, 5H); ¹³C NMR (125 MHz, CDCl₃) δ C_(c)) 20.8, Cb) 23.5, C_(a)) 25.9,C_(d)) 26.1, C_(e)) 36.2, C_(f)) 46.2, C_(g)) 55.8, C_(h)) 61.6, C_(i))64.2, C_(j)) 67.0, C_(k)) 68.2, C_(l)) 125.0, 128.0, 128.8, 128.9 &134.1, C_(n)) 137.9, C_(m)) 167.0; IR (neat) 2954 (s), 2360 (s), 2338(s), 1740 (s), 1455 (s), 1389 (s), 1197 (s), 1141 (s); cm⁻¹; HRMS m/zcalculated for C₁₉H₃₀N₂O₂ (M+H) 319.2386, found 319.2392.

L-Lactyl-Pro Ψ(NHCH₂) N-Methyl-Benzyl-D-Leucinate (compound 50 a).

Compound 44 (20 milligrams, 0.063 millimoles) was dissolved in CH₂Cl₂(0.5 milliliters), and lactic acid (5.55 milligrams, 0.063 millimoles)was added at 0° C., followed by the addition of BOP (28 milligrams,0.063 millimoles and NMM (0.035 milliliters, 0.31 millimoles). Thereaction mixture was stirred at 0° C. and monitored by TLC. When thereaction was completed, the reaction mixture was diluted with ether. Theorganic layer was washed with 5% HCl, 5% NaHCO₃, and brine. Theresulting solution was dried over Na₂SO₄ and concentrated. The crudemixture was purified by column chromatography, eluting with 30%acetone/hexane to afford compound 50 a in 61% yield. The followinganalytical data were obtained for compound 50 a: R_(f) 0.50 (50%Acetone/Hexane); [α]_(D) ²⁵ +21.0 (c=0.2, CHCl₃); ¹H NMR (500 MHz,CDCl₃) δ H_(a)) 0.82-0.92 (d, 6H), H_(b)) 1.20 (d, 3H), H_(c)) 1.45-1.66(m, 2H), H_(d)) 1.66-1.72 (m, 1H), H_(e)) & H_(f)) 1.76-1.90 (m, 4H),H_(g)) & H_(h)) 2.25-2.45 (m, 5H), H_(i)), H_(j)) & H_(k)) 3.19-3.85 (m,4H), H_(l)) 4.10-4.21 (rm, 1H), H_(m)) 5.05-5.18 (s, 2H), H_(n))7.12-7.38 (m, 5H); ¹³C NMR (125 MHz, CDCl₃) δ C_(a)) 22.0, C_(c)) 22.8,C_(d)) 24.2, C_(e)) 24.8, C_(b)) 25.4, C_(f)) 28.1, C_(g)) 37.8, C_(h))38.6, C_(j)) 45.8, C_(i)) 46.2, C_(k)) 56.0, C_(l)) 56.8, C_(m)) 66.0,C_(n)) 128.8, C_(o)) 136.1, C_(p)) 173.8, C_(q)) 174.1; IR (neat), 3415(br), 2955 (s), 2869 (m), 1729 (s), 1638 (s), 1455 (m), 1379 (m), 1366(m), 1147 (m), 1126 (m); HRMS m/z calculated for C₂₂H₃₄N₂O₄ (M+Na)413.2416, found 413.2423.

N-Methyl-leu Ψ(NHCH₂) lac-pro acid (compound 51 a).

Compound 50 a (20 milligrams, 0.063 millimoles) was dissolved in 0.5milliliters MeOH/EtOAc (1:1). The mixture was added to a solution ofMeOH and EtOH (1:1) containing Pd/C catalyst (10 milligrams). Thereaction mixture was shaken in a Parr hydrogenator under H₂ (40 poundsper square inch, gauge) and monitored by TLC. When the reaction wascompleted, the catalyst was removed by filtration. The remainingsolution was concentrated in vacuo. The crude product (compound 51 a)was used directly in the next step. The following analytical data wereobtained for compound 51 a: R_(f) 0.20 (10% MeOH/CH₂Cl₂); [α]_(D) ²⁵+65.0 (c=0.2, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ H_(a)) 0.82-0.92 (d,6H), H_(b)) 1.34 (d, 3H), H_(c)) 1.45-1.55 (m, 1H), H_(d)) 1.78-1.91 (m,2H), H_(e)) 1.91-2.0 (m, 2H), H_(f)) 2.0-2.18 (m, 2H), H_(g)) 2.81 (s,3H), H_(h)) 3.03-3.18 (d, 2H), H_(i)) 3.45-3.54 (m, 1H), H_(j))3.56-3.68 (t, 2H), H_(k)) 4.25-4.35 (m, 1H), H_(l)) 4.40-4.49 (m, 1H);¹³C NMR (125 MHz, CDCl₃) δ C_(a)) 21.0, C_(b)) 22.8, C_(c)) 23.4, C_(d))24.8, C_(e)) 25.8, C_(f)) 29.4, C_(g)) 36.5, C_(h)) 38.6, C_(i)) 47.2,C_(j)) 55.2, C_(k)) 66.4, C_(l)) 68.2, C_(m)) 172.4, C_(n)) 176.2; IR(neat), 3336 (br), 2957 (s), 2870 (m), 1718 (m), 1627 (s), 1466 (s),1368 (s), 1250 (m), 1197 (w), 1128 (w); HRMS m/z calculated forC₁₅H₂₇N₂O₄ (M+Na) 323.1947, found 323.1939.

Cyclo[N-(N-3,4-didehydro-L-prolyl-N-methyl-D-leucyl)-O[[N-[(2S,3S,4S)-4-[(3S,4R,5S)-4-amino-3-hydroxy-5-methyl-heptanoyl]oxy-3-oxo-2,5-dimethylhexanoyl]-L-leucyl]-L-prolyl-N,O-dimethyl-L-tyrosyl]-L-threonyl](compound 72 a).

The crude acid (12.5 milligrams, 0.038 millimoles) was combined with themacrocycle HCl salt (15.0 milligrams, 0.019 millimole) in CH₂Cl₂ (0.25milliliter) at 0° C. HATU (8.2 milligrams, 0.020 millimole) and DIEA(0.026 milliliter, 4 equivalents) was added. The reaction was stirred at0° C. overnight and monitored by TLC. When the reaction was complete,the mixture was diluted with Et₂O and the organic layer was washed with5% HCl, 5% NaHCO₃, and brine. The resulting solution was dried overNa₂SO₄ and concentrated. The crude residue was purified by columnchromatography eluting with 5% MeOH/CH₂Cl₂ to yield the desired product(compound 72 a) in 72% yield. The following analytical data wereobtained for compound 72 a: R_(f) 0.40 (10% MeOH/CH₂Cl₂); [α]_(D) ²⁵+81.2 (c=0.15, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ H_(a)) 0.85-0.97 (m,24H), H_(b)) 1.17-1.27 (m, 2H), H_(c)) 1.28-1.39 (s, 3H), H_(d)) 1.40(d, 3H), H_(e)) 1.43 (d, 3H), H_(f)) 1.46-1.50 (m, 1H) and 1.61 (t, 1H),H_(g)) 1.68-1.73 (m, 1H), H_(h)) 1.74-1.79 (m, 1H), H_(i)) 1.82-1.88 (m,2H), 2.02-2.08 (m, 1H) and 2.11-2.17 (m, 2H), H_(j)) 2.34-2.37 (m, 1H),H_(k)) 2.57 (s, 3H), H_(l)) 2.38 (d, 2H), H_(m)) 3.18 and 3.39 (dd, 1H),H_(n)) 2.83 (s, 3H), H_(o)) 3.37-3.57 (m, 2H), H_(p)) 3.60 (d, 1H),H_(q)) 3.70-3.73 (m, 1H), H_(r)) 3.80 (s, 3H), H_(s)) 4.05-4.14 (m, 3H)and 4.36-4.48 (m, 2H), H_(t)) 4.27 (q, 1H), H_(u)) 4.56 (dd, 1H), H_(v))4.65 (dd, 1H), H_(w)) 4.81 (t, 1H), H_(x)) 5.19 (d, 1H), H_(y)) 5.35(dd, 1H), H_(z)) 5.42 (dd, 1H), H_(aa)) 5.61 (dd, 1H), H_(bb)) 5.73-5.75(m, 1H), H_(cc)) 6.11 (dd, 1H), H_(dd)) 6.85 (d, 2H), H_(ee)) 7.08 (d,2H), H_(ff)) 7.20 (d, 1H), H_(gg)) 7.54 (d, 1H), H_(hh)) 7.78 (d, 1H);¹³CNMR (125 MHz, CDCl₃) δ C_(a)), C_(c)) 11.7, 14.7, 15.2, 16.3, 16.9,18.6, 20.1 and 20.9, C_(d)) 23.3, C_(b)) 23.7, C_(b)) 24.8 and 27.2,C_(i)) 24.9,25.0 and 33.9, C_(f)) 27.9, C_(g)) 31.2, C_(h)) 31.3, C_(j))33.9, C_(l)) 36.2, C_(m)) 38.7, C_(k)) 38.8, C_(n)) 41.3, C_(o)) 46.9,C_(q)) 49.46, C_(s)) 49.52, 53.0, and 67.9, C_(r)) 55.3, C_(v)) 55.4,C_(w)) 55.5, C_(u)) 57.2, C_(y)) 57.6, C_(t)) 63.9, C_(aa)) 65.9, C_(s))66.4, C_(p)) 66.5, C_(x)) 70.4, C_(z)) 81.5, C_(dd)) 114.1, C_(cc))124.0, C_(bb)) 129.1, C_(ii)) 130.0, C_(ee)) 130.3, C_(jj)) 158.6,C_(kk)) 168.6, 169.3, 170.5, 170.6, 171.3, 171.5, 172.4, 173.9, C_(ll))204.9; IR (neat) 3331 (br), 2956 (s), 2871 (m), 1732 (s), 1638.3 (br,overlap), 1543 (m), 1513 (s), 1448 (m), 1379 (m), 1247 (w), 1167 (w)cm⁻¹; HRMS m/z calculated for C₅₇H₉₁N₇O₁₄ (M+H) 1098.6702, found1098.6726 .

Example 2

Synthesis of a 3,4-Dehydro-Proline Side Chain Moiety and Coupling to aDidemnin Macrocycle

Synthesis of the 3,4-dehydroproline unit began withtrans-4-hydroxy-L-proline (compound 52), which was produced as described(Rueger et al., 1982, Can. J. Chem. 60:2918; see FIG. 6). The acid wasfirst protected as its ethyl ester. The amino group was furtherprotected using Boc₂O to yield compound 53. The hydroxyl group wasmesylated using MsCl and pyridine. The mesylate (i.e., methyl sulfonatemoiety of compound 53) was displaced by sodium benzene selenide withinversion of stereochemistry to yield compound 54. Oxidative eliminationof the phenyl selenium group afforded the corresponding alkene (compound55) in 73% yield.

If compound 54 was directly exposed to the basic elimination condition,two regioisomers would be generated. But oxidative elimination ofcompound 54 through phenyl selenium as the intermediate gave only thedesired regioisomer. This regioselectivity may be due to the transitionstate leading to the undesired isomer has larger dipole moment withhigher energy.

Hydrolysis of the ethyl ester (compound 55) yielded the freedehydroproline acid (compound 56), which was coupled with D-leucineester to yield compound 57. Hydrolysis of the methyl ester afforded thefree acid, which was coupled to the didemnin macrocycle salt using HATUto afford compound 58. The Boc protecting group was removed using HClgas, and the HCl salt was neutralized using saturated NaHCO₃ solution toafford the final analog, compound 59.

The steps in this synthesis are now described in greater detail.

Ethyl N-Boc-trans-4-Hydroxyprolinate (compound 53). Ethyltrans-4-hydroxy prolinate hydrochloride salt (1.0 gram, 0.005 mole) wasdissolved in saturated CH₂Cl₂ (10 milliliter). Et₃N (2.09 milliliter,0.015 mole) was added at 0° C., followed by addition of Boc₂O (2.23 g,0.01 mole). The reaction mixture was stirred overnight. When thereaction was complete, the pH was measured, and was then 8. The reactionmixture was washed with ether, and the ether layer was discarded. Theaqueous layer was acidified with 1 normal KHSO₄ to pH 4, followed byextraction three times with ethyl acetate. The organic extracts werecombined, washed with brine, dried, and concentrated. The crude mixturewas purified by column chromatography, eluting with 20% acetone/hexaneto affect the desired product (compound 53) in 71% yield. The followinganalytical data were obtained for compound 53: R_(f) 0.50 (40%Acetone/Hexane); [α]_(D) ²⁵ +71.3 (c=0.2, CHCl₃); ¹H NMR (500 MHz,CDCl₃) δ H_(a)) 1.07-1.13 (t, 6H), H_(b)) 1.32-1.36 (m, 9H), H_(c))1.84-2.15 (m, dr, 2H), H_(d)) 3.23-3.49 (m, dr, 2H), H_(e)) 3.78-3.95(br, 1H), H_(f)) 3.96-4.09 (m, 2H), H_(g)) 4.17-4.26 (m, 1H), H_(h))4.27-4.32 (t, 1H); ¹³C NMR (125 MHz, CDCl₃) δ C_(a)) 14.0, C_(b)) 28.1,C_(c)) 38.2, C_(d)) 54.2, C_(f)) 57.7, C_(g)) 61.0, C_(h)) 69.0, C_(i))80.0, C_(j)) 153.9, C_(k)) 172.6; IR (neat) 3448 (br), 2978 (s), 2935(m), 1746 (m), 1702 (s), 1676 (s), 1477 (m), 1402(s), 1367 (m), 1339 (m)cm⁻¹; HRMS m/z calculated for C₁₁H₂₃N₄O₂ (M+H) 260.1497, found 260.1503.

Ethyl N-Boc-cis-4-phenylselenyl-L-prolinate (compound 54). Sodiumborohydride (0.15 gram, 0.004 mole) was added in small portions, at roomtemperature, to a solution of diphenyl diselenide (0.556 gram, 0.0018mole) in EtOH. The mixture was stirred for about 5 minutes, until thebright yellow color disappeared. The previously prepared mesylate (1.0gram, 0.003 mole) was added, the solution was refluxed for 2 hours, andthe solvent was removed in vacuo. The residue was diluted with Et₂O (5milliliters), and the organic layer was washed with H₂O (10 milliliters)and brine. The resulting organic layer was dried and concentrated. Thecrude oil was purified by column chromatography, eluting with 10%acetone/hexane to afford the product (compound 54) in 85% yield. Thefollowing analytical data were obtained for compound 54: R_(f) 0.53 (30%Acetone/Hexane); [α]_(D) ²⁵ −16.4 (c=0.3, CHCl₃); ¹H NMR (500 MHz,CDCl₃) δ H_(a)) 1.21-1.24 (t, 3H), H_(b)) 1.45 (s, 9H), H_(c)) 2.05 &2.68 (dr, 2H), H_(d)) 3.40.(m, 1H), H_(e)) 3.58 (m, 1H), H_(f1)) 3.95(m, 1H), H_(f2)) & H_(g)) 4.10-4.28 (m, 2H), H_(h)) 7.25, 7.55 (m, 5H);¹³C NMR (125 MHz, CDCl₃) δ C_(a)) 14.0, C_(b)) 28.2, C_(c)) 36.1, C_(d))39.8, C_(e)) 53.6, C_(f)) 58.8, C_(g)) 60.8, C_(i)) 80.2, C_(h)) 127.0,128.2, 134.3, C_(j)) 152.8, C_(k)) 171.8; IR (neat) 2977.0, 1747.1,1701.9, 1477.4, 1394.4, 1190.1, 1114.1 (m) cm⁻¹; HRMS m/z calculated forC₁₈H₂₅NO₄Se (M+H) 399.0948, found 399.0957.

Ethyl N-Boc-3,4-Dehydro-L-prolinate (compound 55). A mixture of compound54 (0.9 gram, 2.26 millimoles) and CH₂Cl₂ was initially cooled to 0° C.in an ice bath. Pyridine (0.27 milliliter, 3.4 millimoles) was addeddropwise to this solution. A solution of 30% aqueous H₂O₂ (0.58milliliter) was then gradually added over a 5 minute period. Thereaction was stirred at room temperature for 1 hour, and then dilutedwith EtOAc. The organic layer was washed with 5% HCl, saturated Na₂CO₃solution, and brine. The resulting solution was dried and concentrated.The residue was purified by column chromatography to afford the desiredproduct (compound 55) in 73% yield. The following analytical data wereobtained for compound 55: R_(f) 0.63 (30% Acetone/Hexane); [α]_(D) ²⁵−32.3 (c=0.3, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ H_(a)) 1.15-1.30 (t,3H), H_(b)) 1.45 (s, 9H), H_(c)) & H_(d)) 4.15-4.30 (m, 4H), H_(e)) 4.98(d, 1H), H_(f)) 5.75 (dt, 1H), H_(g)) 5.95 (dd, 1H); ¹³C NMR (125 MHz,CDCl₃) δ C_(a)) 14.1, C_(b)) 29.2, C_(c)) 53.8, C_(d)) 61.8, C_(e))67.0, C_(h)) 80.1, C_(f)) 125.0, C_(g)) 129.2, C_(i)) 154.0, C_(j))170.2; IR (neat) 3458 (br), 2979 (s), 1786 (s), 1750 (s), 1710 (s), 1448(m), 1395 (m), 1369 (s), 1318 (s), 1258 (m), 1159 (s), 1896 (m) cm⁻¹;HRMS m/z calculated for C₁₂H₁₉NO₄ (M+H) 242.1392, found 242.1386.

N-Boc-3,4-Dehydro-L-proline (compound 56). Ethyl ester 55 (0.18 gram,0.8 millimole) was dissolved in THF/H₂O (1:1, 10 milliliters). LiOH.H₂O(0.33 gram, 8 millimoles) was added to the solution at 0° C. The mixturewas stirred for about 6 hours and monitored by TLC. When the reactionwas completed, THF was removed in vacuo. Saturated NaHCO₃ solution wasadded and washed with ether twice. All aqueous layers were combined andacidified to pH 4 with 1 normal KHSO₄. Ethyl acetate was used to extractthe acidified aqueous solution three times. The extracts were dried andconcentrated to afford the desired acid (compound 56) in 92% yield. Thefollowing analytical data were obtained for compound 56: R_(f) 0.20 (30%acetone/hexane); [α]_(D) ²⁵ +8.4 (c=0.2, CHCl₃); ¹H NMR (500 MHz, CDCl₃)δ H_(a)) 1.49 (s, rm, 9H), H_(b)) 4.25 (d, 2H), H_(c)) 5.05 (d, 1H),H_(d)) 5.80 (dt, 1H), H_(e)) 6.01 (dd, 1H), H_(f)) 8.40 (br, 1H); ¹³CNMR (125 MHz, CDCl₃) δ C_(a)) 28.9, C_(b)) 53.9, C_(c)) 66.2, C_(g))81.8, C_(d)) 124.2, C_(e)) 129.9, C_(h)) 154.5, C_(i)) 165.3; IR (neat)3000-3400 (br), 2957 (s), 1736 (s), 1704 (s), 1666 (s), 1400.2 (s), 1367(m), 1177 (s), 1136 (s) cm⁻¹; HRMS m/z calculated for C₁₀H₁₅NO₄ (M+H)213.1079, found 242.1086.

N-Boc-3,4-Dehydro-L-Prolyl-Methyl-N-Methyl-Leucinate (compound 57).Compound 56 (0.1 gram, 0.469 millimole) was dissolved in CH₂Cl₂ (5milliliters). At 0° C., HATU (0.26 gram, 0.58 millimole) was added,followed by the addition of DIEA (0.26 milliliter, 1.88 millimoles).Finally, dimethyl D-leucine hydrochloride salt (0.09 gram, 0.469millimole) was added to the mixture. The reaction mixture was stirredfor 6 hours and monitored by TLC. When the reaction was completed, themixture was diluted with Et₂O and the organic layer was washed with 5%HCl, 5% NaHCO3, and brine. The resulting solution was dried andconcentrated. The crude residue was purified by column to afford thedesired product (compound 57) in 76% yield. The following analyticaldata were obtained for compound 57: R_(f) 0.32 (30% Acetone/Hexane);[α]_(D) ²⁵ −22.7 (c=0.4, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ H_(a))0.90-1.05 (d, rm, 6H), H_(b)) 1.48 (s, rm, 9H), H_(c)) & H_(d))1.65-1.80 (m, 3H), H_(e)) 3.05 (s, 3H), H_(f)) 3.75 (s, 3H), H_(g))4.20-4.35 (m, 2H), H_(h)) 5.28 (t, 1H), H_(i)) 5.40 (d, 1H), H_(j)) 5.70(dt, 1H), H_(k)) 6.01 (dd, 1H); ¹³C NMR (125 MHz, CDCl₃) δ C_(a)) 22.0,23.8, C_(c)) 25.2, C_(b)) 29.0, C_(d)) 37.8, C_(e)) 52.2, C_(h)) 53.8,C_(i)) 65.8, C_(l)) 80.0, C_(j)) 124.0, C_(k)) 129.2, C_(m)) 159.5,C_(n)) 171.0, C_(o)) 172.2; IR (neat) 2956 (s), 1743 (s), 1705 (s),1667(s), 1400(s), 1366(m), 1316(w), 1258 (w), 1177 (m), 1128 (m) cm⁻¹;HRMS m/z calculated for C₁₈H₃₀N₂O₅ (M+H) 355.2233, found 355.2225.

N-Boc-3,4-Dehydro-L-Prolyl-N-Methyl-Leucine (compound 57 a). Compound 57(0.13 gram, 0.37 millimole) was dissolved in THF/H₂O (1:1, 5milliliters). LiOH.H₂O (0.15 gram, 3.7 millimoles) was added to thesolution at 0° C. The mixture was stirred for about 6 hours andmonitored by TLC. When the reaction was completed, THF was removed invacuo. Saturated NaHCO₃ solution was added and washed with ether twice.All aqueous layers were combined and acidified to pH 4 with 1 normalKHSO₄. Ethyl acetate was used to extract the acidified aqueous solutionthree times. The extracts were dried and concentrated to afford thedesired acid (compound 57 a) in 92% yield. The following analytical datawere obtained for compound 57 a: R_(f) 0.20 (40% Acetone/Hexane);[α]_(D) ²⁵ +52.3 (c=0.2, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ H_(a))0.85-1.05 (d, rm, 6H), H_(b)) 1.48 (s, rm, 9H), H_(c)) & H_(d))1.65-1.92 (m, 3H), H_(e)) 3.12 (s, rm, 3H), H_(f)) br, 1H, H_(g))4.10-4.28 (m, 2H), H_(h)) 5.15 (t, 1H), H_(i)) 5.35 (d, 1H), H_(j)) 5.70(dt, 1H), H_(k)) 6.01 (dd, 1H); ¹³C NMR (125 MHz, CDCl₃) δ C_(a)) 23.3,24.5, C_(c)) 25.0, C_(b)) 28.3, C_(d)) 31.5, C_(g)) 38.5, C_(e)) 53.6,C_(i)) 55.8, C_(h)) 65.2, C_(l)) 80.0, C_(j)) 124.1, C_(k)) 129.5,C_(m)) 153.8, C_(n)) 171.3, C_(o)) 175.3; IR (neat) 3300 (br), 2957 (s),1731 (m), 1783 (s), 1667 (s), 1612 (m), 1481 (m), 1400 (s), 1367 (m),1177 (s), 1136.7 (s) cm⁻¹; HRMS m/z calculated for C₁₇H₂₈N₂O₅ (M+H)341.2076, found 341.2063.

Cyclo[N-(N-Boc-3,4-dehydro-L-prolyl-N-methyl-D-leucyl)-O[[N-[(2S,3S,4S)-4[(3S,4R,5S)-4-amino-3-hydroxy-5-methyl-heptanoyl]oxy-3-oxo-2,5-dimethylhexanoyl]-L-leucyl]-L-prolyl-N,O-dimethyl-L-tyrosyl]-L-threonyl](compound 58). The crude acid 57 a (5.0 milligrams, 0.012 millimole) wascombined with the macrocycle HCl salt (14.9 milligrams, 0.012 millimole)in CH₂Cl₂ (0.1 milliliter) at 0° C. HATU (48 milligrams, 0.012millimole) and DIEA (0.048 milliliter, 0.048 millimole) was added. Thereaction was stirred at 0° C. overnight and monitored by TLC. When thereaction was complete, the mixture was diluted with Et₂O and the organiclayer was washed with 5% HCl, 5% NaHCO₃, and brine. The resultingsolution was dried and concentrated. The crude residue was purified bycolumn chromatography, eluting with 5% MeOH/CH₂Cl₂ to afford the desiredproduct (compound 58) in 72% yield. The following analytical data wereobtained for compound 58: R_(f) 0.40 (10% MeOH/CH₂Cl₂); [α]_(D) ²⁵ −83.2(c=0.3, CHCl₃); 1H NMR (500 MHz, CDCl₃) δ H_(a)) 0.85-0.97 (m, 24H),H_(b)) 1.17-1.27 (m, 3H), H_(c)) 1.45 (s, 9H), H_(d)) 1.40 (d, 3H),H_(e)) 1.43 (d, 3H), H_(f)) 1.46-1.50 (m, 1H) and 1.61 (t, 1H), H_(g))1.68-1.73 (m, 1H), H_(h)) 1.74-1.79 (m, 1H), H_(i)) 1.82-1.88 (m, 2H),2.02-2.08 (m, 1H) and 2.11-2.17 (m, 2H), H_(j)) 2.34-2.37 (m, 1H),H_(k)) 2.57 (s, 3H), H_(l)) 2.63 (dd, 1H) and 2.95 (d, 1H), H_(m)) 3.18and 3.39 (dd, 1H), H_(n)) 3.23 (s, 3H), H_(o)) 3.57-3.61 (m, 2H), H_(p))3.33 (d, 1H), H_(q)) 3.69-3.71 (m, 1H), H_(r)) 3.80 (s, 3H), H_(s))4.05-4.14 (m, 2H) and 4.36-4.48 (m, 2H), H_(t)) 4.27 (q, 1H), H_(u))4.56 (dd, 1H), H_(v)) 4.65 (dd, 1H), H_(w)) 4.81 (t, 1H), H_(x)) 5.19(d, 1H), H_(y)) 5.35 (dd, 1H), H_(z)) 5.42 (dd, 1H), H_(aa)) 5.61 (dd,₁H), H_(bb)) 5.73-5.75 (m, 1H), H_(cc)) 6.11 (dd, 1H), H_(dd)) 6.85 (d,2H), H_(ee)) 7.08 (d, 2H), H_(ff)) 7.20 (d, 1H), H_(gg)) 7.54 (d, 1H),H_(hh)) 7.78 (d, 1H); ¹³C NMR (125 MHz, CDCl₃) δ C_(a)) 11.7, 14.7,15.2, 16.3, 16.9, 18.6, 20.1 and 20.9, C_(d)) 23.3, C_(e)) 23.7, C_(a))24.8 and 27.2, C_(i)) 24.9, 25.0 and 33.9, C_(f)) 27.9, C_(c)) 28.6,C_(g)) 31.2, C_(h)) 31.3, C_(j)) 33.9, C_(l)) 36.2, C_(m)) 38.7C_(k))38.8, C_(n)) 41.3, C_(o)) 46.9, C_(q)) 49.46, C_(s)) 49.52, 53.0, and67.9, C_(r)) 55.3, C_(v)) 55.4, C_(w)) 55.5, C_(u)) 57.2, C_(y)) 57.6,C_(t)) 63.9, C_(aa)) 65.9, C_(p)) 66.5, C_(x)) 70.4, C_(z)) 81.5, C_(z))81.7, C_(dd)) 114.1, C_(cc)) 124.0, C_(bb)) 129.1, C_(ii)) 130.0,C_(ee)) 130.3, C_(jj)) 158.6, C_(kk)) 168.6, 169.3, 170.5, 170.6, 171.3,171.5, 172.4, 173.9, 204.9; IR (neat) 3337 (s), 2959 (s), 2870 (m), 1733(s), 1645 (s), 1640 (s), 1543 (m), 1514 (m), 1454 (m), 1407 (m), 1368(m), 1302 (w), 1248 (m), 1171 (m) cm⁻¹; HRMS m/z calculated forC₅₉H₉₁N₇O₁₅ (M+Na) 1160.6471, found 1160.6415.

Cyclo[N-(N-3,4-dehydro-L-prolyl-N-methyl-D-leucyl)-O[[N-[(2S,3S,4S)-4[(3S,4R,5S)-4-amino-3-hydroxy-5-methyl-heptanoyl]oxy-3-oxo-2,5-dimethylhexanoyl]-L-leucyl]-L-prolyl-N,O-dimethyl-L-tyrosyl]-L-threonyl](compound 59). Compound 58 (4 milligrams) was dissolved in HCl.dioxane(0.5 milliliter) and stirred at room temperature. When the reaction wascompleted, the solvent was removed in vacuo. Toluene was added twice,and the solution was concentrated. The residue was dried in vacuoovernight to afford the desired HCl salt in 98%. The HCl salt wasdissolved in EtOAc, washed with saturated NaHCO₃, and the organic layerwashed again with brine, dried over Na₂SO₄, and concentrated to affordthe desired product (compound 59) in 75% yield. The following analyticaldata were obtained for compound 59: R_(f) 0.20 (10% MeOH/CH₂Cl₂);[α]_(D) ²⁵ −242.3 (c=0.2, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ H_(a))0.79-0.97 (m, 24H), H_(b)) 1.12-1.23 (m, 3H), H_(d)) 1.32-1.40 (d, 3H),H_(e)) 1.43 (d, 3H), H_(f)) 1.46-1.50 (m, 1H) and 1.61 (t, 1H), H_(g))1.68-1.73 (m, 1H), H_(h)) 1.74-1.79 (m, 1H), H_(i)) & H_(j)) 1.82-1.88(m, 2H), 2.02-2.08 (m, 1H) and 2.11-2.17 (m, 3H), H_(k)) 2.25 (s, 3H),H_(l)) 2.51 (d, 2H), H_(m)) 3.18 and 3.39 (dd, 1H), H_(n)) 2.95 (s, 3H),H_(o)) 3.57-3.61 (m, 2H), H_(p)) 3.33 (d, 1H), H_(q)) 3.69-3.71 (m, 1H),H_(r)) 3.73 (s, 3H), H_(s)) 3.90-4.18 (m, 2H), H_(t)) 4.49 (q, 1H),H_(u)) 4.56 (dd, 1H), H_(v)) 4.65 (dd, 1H), H_(w)) 4.81 (t, 1H), H_(x))5.19 (m, 3H), H_(bb)) & H_(aa)) 5.73-5.75 (m, 3H), H_(cc)) & H_(z))6.01(dd, 3H), H_(dd)) 6.85 (d, 2H), H_(ee)) 7.08 (d, 2H), H_(ff)) 7.20 (d,1H), H_(gg)) 7.54 (d, 1H), H_(hh)) 7.78 (d, 1H); ¹³C NMR (125 MHz,CDCl₃) δ C_(a)) 11.7, 14.7, 15.2, 16.3, 16.9, 18.6, 20.1 and 20.9,C_(d)) 23.3, C_(e)) 23.7, C_(a)) 24.8 and 27.2, C_(i)) 24.9, 25.0 and33.9, C_(f)) 27.9, C_(c)) 28.6, C_(g)) 31.2, C_(h)) 31.3, C_(j)) 33.9,C_(l)) 36.2, C_(m)) 38.7, C_(k)) 38.8, C_(n)) 41.3, C_(o)) 46.9, C_(q))49.46, C_(s)) 49.52, 53.0, and 67.9, C_(r)) 55.3, C_(v)) 55.4, C_(w))55.5, C_(u)) 57.2, C_(y)) 57.6, C_(t)) 63.9, C_(aa)) 65.9, C_(p)) 66.5,C_(x)) 70.4, C_(z)) 81.5, C_(dd)) 114.1, C_(cc)) 124.0, C_(bb)) 129.1,C_(ii)) 130.0, C_(ee)) 130.3, C_(jj)) 158.6, C_(kk)) 168.6, 169.3,170.5, 171.3, 171.5, 172.4, 173.9, C_(ll)) 204.9; IR (neat) 3337 (s),2958 (s), 2861 (m), 1734 (s), 1642 (s), 1638 (s), 1547 (m), 1514 (m),1451 (m), 1385 (w), 1243 (w), 1166 (w) cm⁻¹; HRMS m/z calculated forC₅₄H₈₃N₇O₁₃ (M+H) 1038.6127, found 1038.6103.

The disclosure of every patent, patent application, and publicationcited herein is hereby incorporated herein by reference in its entirety.

While this invention has been disclosed with reference to specificembodiments, other embodiments and variations of this invention can bedevised by others skilled in the art without departing from the truespirit and scope of the invention. The appended claims include all suchembodiments and equivalent variations.

1. A composition comprising a tamandarin analog having the structure

wherein: i) R¹ is selected from the group consisting of—(N-methyl)leucine-deoxo-pro line,—(N-methyl)leucine-deoxo-proline-lactate,—(N-methyl)leucine-deoxo-proline-pyruvate,—(N-methyl)leucine-deoxo-proline-lactate-(a first fluorophore),—(N-methyl)leucine-deoxo-proline-lactate-glutamine-pyroglutamate,—(N-methyl)leucine-deoxo-proline-lactate-glutamine-cyclopentanoate,—(N-methyl)leucine-deoxo-proline-alanine-leucine-pyroglutamate, and—(N-methyl)leucine-deoxo-proline-(N-methyl-alanine)-leucine-pyroglutamate;—(N-methyl)leucine-dehydro-proline,—(N-methyl)leucine-dehydro-proline-plactate,—(N-methyl)leucine-dehydro-proline-pyruvate,—(N-methyl)leucine-dehydro-proline-lactate-(a first fluorophore),—(N-methyl)leucine-dehydro-proline-lactate-glutamine-pyroglutamate,—(N-methyl)leucine-dehydro-proline-lactate-glutamine-cyclopentanoate,—(N-methyl)leucine-dehydro-proline-alanine-leucine-pyroglutamate, and—(N-methyl)leucine-dehydro-prol2ne-(N-methyl-alanine)-leucine-pyroglutamate;ii) R² and R³ are one of (a) R² is selected from the group consisting ofan isoleucine side chain, a valine side chain, an alanine side chain, anorleucine side chain, a norvaline side chain, a leucine side chain, ahistidine side chain, a tryptophan side chain, an arginine side chain, alysine side chain, a second fluorophore, and a substituent having thestructure

and R³ is selected from the group consisting of —CH₃ and —H; or (b) R²and R³ form the structure

iii) each of R⁵, R⁶, R⁷, R⁸, and R⁹, when present, is independentlyselected from the group consisting of —H, —OH, —OCH₃, —CO(C₆H₅), —Br,—I, —F, —Cl, —CH₃, and —C₂H₅; iv) R⁴ is selected from the groupconsisting of an isoleucine side chain and a valine side chain; v) X isselected from the group consisting of —O— and —(NH)—; vi) Y is selectedfrom the group consisting of —H and a hydroxyl protecting group; vii)R¹⁰ is selected from the group consisting of a leucine side chain and alysine side chain; and viii) the molecule is not tamandarin A. 2-55.(canceled)